Humanized c-Kit Antibody

ABSTRACT

This invention relates to compositions and methods for treating c-Kit associated disorders such as fibrosis, and more particularly, to compositions containing humanized c-Kit antibodie

This application is a divisional application of U.S. patent applicationSer. No. 13/046,463 filed Mar. 11, 2011, which is a continuationapplication of U.S. Pat. No. 7,915,391 issued Mar. 21, 2011, whichclaims the benefit of U.S. Provisional Application No. 60/794,771, filedApr. 24, 2006. The disclosures of all these applications are herebyincorporated by reference in their entireties herein.

TECHNICAL FIELD

This invention relates to compositions for treating c-Kit associatedinflammatory, fibrotic, autoimmune and cancerous diseases and tocompositions containing humanized c-Kit antibodies.

BACKGROUND OF THE INVENTION

Mast cells have been implicated in the mediation of inflammatoryconditions such as asthma, rheumatoid arthritis and inflammatory boweldisease and the role in allergic inflammation is widely recognized. Mastcells are increased in number in lung explants from severe asthmaticsand are the major source of clinically relevant inflammatory mediatorssuch as leukotriences, histamine and Th2 cytokines. Mast cells are themajor source of pre-formed TNF in disease tissues.

Stem Cell Factor (SCF) is a glycoprotein that signals through the celland membrane associated tyrosine kinase receptor hereafter defined asc-Kit, and this signaling pathway plays a key role in hematopoiesisacting both as a positive and negative regulator, often in synergy withother cytokines. A soluble shed c-Kit receptor may play a role inregulating SCF. C-Kit is expressed on pluripotent hematopoietic stemcells which are the precurors to mature cells belonging to lymphoid anderythroid lineages. Unlike other hematopoietic cells, mast cellprecursors and mature mast cells retain high-levels of c-Kit expression.Hence SCF signaling via c-Kit is vital for mast cell development,function, trafficking and survival. It also plays a role ingametogenesis, and melanogenesis. Mice with inactivating c-Kit mutationsin the W locus have virtually no mast cells. Activating c-Kit mutationsin man are associated with mastocytosis.

c-Kit positive pluripotent hematopoietic stem cells are precursors tomultiple cell types including mesenchymal cells, fibroblasts and mastcells. Fibrotic disease is characterized in part by excessive fibroblastactivity and proliferation resulting in the extracellular matrixdeposition. C-Kit positive bone marrow pluripotent hematopoietic stemcells have been reported to be a source of the fibroblasts and mastcells in fibrotic tissues.

Mast cells can provide a sustained source of inflammatory, angiogenic,mitogenic and fibrogenic mediators. Mast cells are functionally andanatomically coupled to fibroblasts and have a direct role in activatingfibroblasts. Mast cells increase the kinetics and magnitude offibroblast mediated collagen contraction, extracellular matrixdeposition and can transform fibroblasts into myofibroblasts.Fibroblasts in turn secrete SCF to further activate and expand mastcells, and both cell types are components of the fibrogenic network.

Mast cell number and mast cell mediators are significantly elevated inmost human fibrotic diseases including idiopathic pulmonary fibrosis(IPF) and Scleroderma. Differential mast cell phenotypes are detected insome scleroderma patients and in the Tsk mouse model of scleroderma. Anaggressive systemic form of mastocytosis may be characterized bymyelofibrosis indicating that mast cells can be effector cells infibrosis.

Gleevec™ and others in the class like Sutent™ are multi-targetedtyrosine kinase receptor inhibitors that can target c-Kit signalingactivity, but inhibit a number of other kinases. These kinase inhibitorsare indicated for the oncology setting. Myelosuppression, anemia and anumber of side-effects including cardiotoxicity and peripheral edemahave been reported for Gleevec. Therefore these molecules may notpossess the best benefit to risk profile for chronic treatment ofdiseases associated with c-Kit signaling. Thus, there is a need for newtherapies and reagents, particularly those that are more potent andselective, and possess better safety profiles for the treatment of c-Kitassociated inflammatory and fibrotic diseases. Such a compound couldalso show significantly better efficacy and safety profiles in oncologicdiseases such as myeloid derived leukemia, diseases associated withc-Kit mutations such as GIST and mastocytosis, diseases associated withover-expression of c-kit and/or excessive SCF autocrine activity as inmelanoma and various SCLCs. Therapies and reagents targeting human c-Kitand capable of affecting a therapeutic benefit without significantadverse effects are currently lacking.

SUMMARY OF THE INVENTION

The invention provides agents that are antagonists and neutralantagonists of SCF at the cell-associated and membrane c-Kit receptor,such as monoclonal antibodies. In a more specific embodiment, humanized(non-murine) monoclonal antibodies that bind c-Kit are provided. In yetmore specific embodiments, the humanized antibodies of the inventioncomprise an amino acid sequence selected from those set forth in SEQ IDNOs 2, 4, and 6. The invention also provides nucleic acids encoding anyof the preceding antibodies or specific binding agents. In a relatedembodiment of the invention, a vector comprising any of theaforementioned nucleic acid sequences is provided. In still anotherembodiment, a host cell is provided comprising any of the aforementionednucleic acids or vectors.

In one embodiment, c-Kit binding agents and neutral antagonists maycomprise an amino acid sequence of SEQ ID NO: 2, 4 or 6. In anotherembodiment, any of the aforementioned agents comprise an amino acidsequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identicalto one or more of the amino acid sequences set forth in SEQ ID NO: 2, 4or 6. In such embodiments, the sequence variation relative to SEQ ID NO:2, 4 or 6, respectively, may represent, for example, a conservativesubstitution of the corresponding framework region of an IgG using analternative human amino acid at that position.

In exemplary embodiments, the antibody or specific binding agent thatbinds c-Kit comprises an amino acid sequence set forth in SEQ ID NO: 2,4, or 6. However, it is contemplated that the antibodies of theinvention may be a mixture of IgG antibody isotypes (for example, amixture of the IgG1, IgG2, IgG3 or IgG4 subtypes).

Any of the aforementioned antibodies may be, e.g., a native or mutatedIgG antibody (for example of the IgG1 or IgG3 subtype, or any other IgGsubtype). The aforementioned antibodies can exhibit an aviditycharacterized by a k_(d) of lower than 1×10⁻², or lower than 1×10⁻³, or1×10⁻⁴, 1×10⁻⁵, 1×10⁻⁶, 1×10⁻⁷, 1×10⁻⁸, or 1×10⁻⁹, as determined bysurface plasmon resonance (BIAcore analysis).

The aforementioned antibodies can exhibit a neutral antagonist IC50 oflower than 1×10⁻², or lower than 1×10⁻³, or 1×10⁻⁴, 1×10⁻⁵, 1×10⁻⁶,1×10⁻⁷, 1×10⁻⁸, or 1×10⁻⁹, as determined by cellular assays. In aparticular embodiment, the affinity and functional potency of thehumanized antibody is at least comparable to the affinity and potency ofa parent murine antibody. In a preferred embodiment, the humanizedantibody is not an agonist of the c-Kit receptor and does not activatemast cells which could lead to anaphylactoid reactions and shouldexhibit a PD/PK and immunogenicity profile that is at least comparableto the parent murine antibody.

Numerous methods are contemplated in the present invention. For example,a method of producing an aforementioned antibody or specific bindingagent is provided comprising culturing the aforementioned host cell suchthat the nucleic acid is expressed to produce the antibody or agent.Such methods may also comprise the step of recovering the antibody oragent from the host cell culture. In a related embodiment, an isolatedantibody or agent produced by the aforementioned method is provided.

The invention further provides methods of using any of the precedingantibodies or specific binding agents, for example, to treat or preventa c-Kit associated disorder by administering an effective amountthereof. One example of such disorder to be treated is a fibroticdisease.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: SR-1 inhibits mast cells in wound activated model.

FIG. 2: Mast cell counts in a wound healing model.

FIG. 3: Humanized SR-1 isoforms inhibit stem cell factor (SCF) inducedactivation of c-Kit and subsequent phosphorylation via c-Kit.

DETAILED DESCRIPTION

The murine anti-human c-Kit antibody SR-1 is described in U.S. Pat. No.5,919,911, and U.S. Pat. No. 5,489,516, each of which are incorporatedherein by reference. SR-1 showed suitable binding properties to c-kitand blocked SCF-mediated receptor signaling, however this molecule didnot possess all the characteristics that would be desired in a humantherapeutic beyond the obvious immunogenicity issues. Broudy reportedthat SR-1 possessed some agonist-like activity that could lead toreceptor internalization and phosphorylation J Cell Physiol. 1994 March;158(3):545-54. These functional activities render the molecule lessefficacious and less safe. Though humanization of monoclonals is anestablished methodology and that biological activities are generallyexpected to be appropriately translated, the conformation of thehumanized SR-1 antibody depending on the human framework may bring aboutdifferent intrinsic activities at c-Kit and thus biological functions.In this particular example, the desired pharmacological properties butnot the undesired “agonistic” properties would be sought, but themethodology to achieve this has not been published. The complementaritydetermining regions of the SR-1 antibody were inserted into a uniquecombination of the human heavy and light chains of structurallydiffering IgG1 and IgG2 and IgG4 while surprisingly maintaining similaraffinity to c-Kit. However, each of these framework regions proved tohave disadvantages.

The humanized SR-1 in the IgG2 background proved to have high affinityfor c-Kit, but in multiple cell types was unable to fully blockSCF-mediated receptor internalization and in cultured mast cell assaysled to c-Kit phosphorylation, a survival signal, and mediated unusualclumping of the cells. These properties are potentially undesirablesince the aim of a therapeutic strategy is to apoptotically deplete mastcells and precurors by blocking the survival SCF signal and to avoidmast cell activation that could lead to anaphylactoid reactions in vivo.When the mouse SR-1 CDR regions were inserted in the human IgG1framework, affinity and functional potency were also maintained, butthis background is less desirable due to the complement activation andcell mediated cytotoxicity often found with this isotype of antibody.When the mouse SR-1 CDR regions were inserted in the human IgG4framework, affinity and functional potency were also maintained, butunexpectedly this molecule showed significant aggregation uponpurification and scaleup.

Thus the present inventors sought to overcome the deficiencies in eachof these molecules by creating an antibody that does not have complementactivation, and does not activate c-kit and mast cells while retainingdesired affinity, neutral antagonist potency for the membrane c-Kitreceptor, and not the soluble c-Kit receptor. This antibody should alsoshow appropriate PD/PK and is efficacious for depleting mast cells andwithout evidence of mast cell agonsim in vivo.

Humanized SR-1 kappa Light Chain

Seq Id No: 1 represents the nucleic acid encoding the SR-1 humanizedkappa light chain.

ATGGTGTTGCAGACCCAGGTCTTCATTTCTCTGTTGCTCTGGATCTCTGGTGCCTACGGGGACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAGAGCCAGTGAAAGTGTTGATATTTATGGCAATAGTTTTATGCACTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACCTTGCATCCAACCTAGAATCTGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAAAATAATGAGGATCCGTACACGTTCGGAGGTGGGACCAAGGTGGAAATAAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGA (SEQ ID NO: 1)

Seq Id No: 2 is as follows where the bold face type represents the CDR's(e.g., CDR1 is amino acids 43 to 58 of SEQ ID NO: 2, CDR2 is amino acids74 to 80 of SEQ ID NO: 2 and CDR3 is amino acids 113 to 121 of SEQ IDNO: 2):

-   -   1 MVLQTQVFIS LLLWISGAYG DIVMTQSPDS LAVSLGERAT INCRASESVD

51 IYGNSFMHWY QQKPGQPPKL LIYLASNLES GVPDRFSGSG SGTDFTLTIS

101 SLQAEDVAVY YCQQNNEDPY TFGGGTKVEI KRTVAAPSVF IFPPSDEQLK 151SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS 201 STLTLSKADYEKHKVYACEV THQGLSSPVT KSFNRGEC (SEQ ID NO:2).

A mature humanized kappa light chain is amino acids 20 to 248 of SEQ IDNO: 2.

Humanized SR-1 Aglyco-IgG1 Heavy Chain Seq Id No: 3

ATGGACTGGACCTGGAGGGTCTTCTGCTTGCTGGCAGTGGCCCCAGGTGCCCACTCCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCAGTTACAATATGCACTGGGTGCGCCAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGTTATTTATTCAGGAAATGGTGATACTTCCTACAATCAGAAGTTCAAAGGCAGGGTCACCATTACCGCTGACAAATCCACCAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGAGAGGGATACTCGTTTTGGTAACTGGGGCCAAGGGACTCTGGTCACCGTCTCTAGTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACCAGAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA (SEQ ID NO:3).

The CDR's are represented in bold face type (e.g., CDR1 is amino acids50 to 54 of SEQ ID NO: 4, CDR2 is amino acids 69 to 85 of SEQ ID NO: 4,and CDR 3 is amino acids 118 to 125 of SEQ ID NO: 4):

-   -   1 MDWTWRVFCL LAVAPGAHSQ VQLVQSGAEV KKPGASVKVS CKASGYTFTS

51 YNMHWVRQAP GQGLEWMGVI YSGNGDTSYN QKFKGRVTIT ADKSTSTAYM

101 ELSSLRSEDT AVYYCARERD TRFGNWGQGT LVTVSSASTK GPSVFPLAPS 151SKSTSGGTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS 201 LSSVVTVPSSSLGTQTYICN VNHKPSNTKV DKKVEPKSCD KTHTCPPCPA 251 PELLGGPSVF LFPPKPKDTLMISRTPEVTC VVVDVSHEDP EVKFNWYVDG 301 VEVHNAKTKP REEQYQSTYR VVSVLTVLHQDWLNGKEYKC KVSNKALPAP 351 IEKTISKAKG QPREPQVYTL PPSRDELTKN QVSLTCLVKGFYPSDIAVEW 401 ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA

451 LHNHYTQKSL SLSPGK (Seq Id No: 4)

Humanized SR-1 IgG2 Heavy Chain

ATGGACTGGACCTGGAGGGTCTTCTGCTTGCTGGCAGTGGCCCCAGGTGCCCACTCCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCAGTTACAATATGCACTGGGTGCGCCAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGTTATTTATTCAGGAAATGGTGATACTTCCTACAATCAGAAGTTCAAAGGCAGGGTCACCATTACCGCTGACAAATCCACCAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGAGAGGGATACTCGTTTTGGTAACTGGGGCCAAGGGACTCTGGTCACCGTCTCTAGTGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCCAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA (Seq Id No: 5)

The following is the full length amino acid sequence of the SR-1 IgG2heavy chain, and the CDR's are represented in bold face type:

-   -   1 MDWTWRVFCL LAVAPGAHSQ VQLVQSGAEV KKPGASVKVS CKASGYTFTS

51 YNMHWVRQAP GQGLEWMGVI YSGNGDTSYN QKFKGRVTIT ADKSTSTAYM

101 ELSSLRSEDT AVYYCARERD TRFGNWGQGT LVTVSSASTK GPSVFPLAPC 151SRSTSESTAA LGCLVKDYFP EPVTVSWNSG ALTSGVHTFP AVLQSSGLYS 201 LSSVVTVPSSNFGTQTYTCN VDHKPSNTKV DKTVERKCCV ECPPCPAPPV 251 AGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSHEDPEVQF NWYVDGVEVH 301 NAKTKPREEQ FNSTFRVVSV LTVVHQDWLNGKEYKCKVSN KGLPAPIEKT 351 ISKTKGQPRE PQVYTLPPSR EEMTKNQVSL TCLVKGFYPSDIAVEWESNG 401 QPENNYKTTP PMLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH

451 YTQKSLSLSP GK (Seq Id No: 6)

SR-1 MULC

ATGGAGACAGACACACTCCTGCTATGGGTGCTGCTGCTCTGGGTTCCAGGTTCCACAGGTAACATTGTGTTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCTGAGGGCCACCATATCCTGCAGAGCCAGTGAAAGTGTTGATATTTATGGCAATAGTTTTATGCACTGGTACCAGCAGAAACCAGGACAGCCACCCAAACTCCTCATCTATCTTGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTGATCCTGTGGAGGCTGATGATGCTGCAACCTATTACTGTCAGCAAAATAATGAGGATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTGA (SEQ ID NO: 7)

The CDR's are represented in bold face type:

-   -   1 METDTLLLWV LLLWVPGSTG NIVLTQSPAS LAVSLGLRAT ISCRASESVD

51 IYGNSFMHWY QQKPGQPPKL LIYLASNLES GVPARFSGSG SRTDFTLTID

101 PVEADDAATY YCQQNNEDPY TFGGGTKLEI K RADAAPTVS IFPPSSEQLT 151SGGASVVCFL NNFYPKDINV KWKIDGSERQ NGVLNSWTDQ DSKDSTYSMS 201 STLTLTKDEYERHNSYTCEA THKTSTSPIV KSFNRNEC(Seq Id No: 8)

SR-1 mulgG2a Heavy ChainATGGGATGGAGTTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCCCAGGTGCAACTGCAGCAGCCTGGGGCTGAGCTGGTGAAGCCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACATTTACCAGTTACAATATGCACTGGGTAAAGCAGACACCTGGACAGGGCCTGGAATGGATTGGAGTTATTTATTCAGGAAATGGTGATACTTCCTACAATCAGAAGTTCAAAGGCAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAAATCAACAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAGAGAGGGATACTCGTTTTGGTAACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGGAGATACAACTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGTGACCTTGACCTGGAACTCTGGATCCCTGTCCAGTGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACCCTCAGCAGCTCAGTGACTGTAACCTCGAGCACCTGGCCCAGCCAGTCCATCACCTGCAATGTGGCCCACCCGGCAAGCAGCACCAAGGTGGACAAGAAAATTGAGCCCAGAGGGCCCACAATCAAGCCCTGTCCTCCATGCAAATGCCCAGCACCTAACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAPAGACCTCCCAGCGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAACAGGTCACTCTGACCTGCATGGTCACAGACTTCATGCCTGAAGACATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAAACTACAAGAACACTGAACCAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGCTACTCctgttcagtggtccacgagggtctgcacaatcaccacacgactaagagcttctcccggactccgggtaaatgA (Seq Id No: 9)

1 MGWSCIILFL VATATGVHSQ VQLQQPGAEL VKPGASVKMS CKASGYTFTS YNMHWVKQTP 61GQGLEWIGVI YSGNGDTSYN QKFKGKATLT ADKSSSTAYM QINSLTSEDS AVYYCARERD 121TRFGNWGQGT LVTVSAAKTT APSVYPLAPV CGDTTGSSVT LGCLVKGYFP EPVTLTWNSG 181SLSSGVHTFP AVLQSDLYTL SSSVTVTSST WPSQSITCNV AHPASSTKVD KKIEPRGPTI 241KPCPPCKCPA PNLLGGPSVF IFPPKIKDVL MISLSPIVTC VVVDVSEDDP DVQISWFVNN 301VEVHTAQTQT HREDYNSTLR VVSALPIQHQ DWMSGKEFKC KVNNKDLPAP IERTISKPKG 361SVRAPQVYVL PPPEEEMTKK QVTLTCMVTD FMPEDIYVEW TNNGKTELNY KNTEPVLDSD 421GSYFMYSKLR VEKKNWVERN SYSCSVVHEG LHNHHTTKSF SRTPGK (Seq Id No: 10)

The light chain CDR1 of SR-1 is RASESVDIYGNSFMH (amino acids 44 to 58 ofSEQ ID NO: 8), CDR2 is LASNLES (amino acids 74 to 80 of SEQ ID NO: 8),and CDR3 is QQNNEDPYT, (amino acids 111 to 121 of SEQ ID NO: 8). Heavychain CDR1 is SYNMH, (amino acids 50 to 54 of SEQ ID NO: 10), CDR2 isVIYSGNGDTSYNQKFKG, (amino acids 69 to 85 of SEQ ID NO: 10), CDR3 isRDTRFGN, (amino acids 118 to 125 of SEQ ID NO: 10).

It is understood that each of the heavy and light chains depicted in thepresent application are processed in the cell to a mature form.Accordingly, signal peptides are cleaved and in the case of heavy chainsof antibodies, the C-terminal lysine is cleaved. Accordingly, the matureform is processed proteolytically and also includes other posttranslational modifications such as glycosylation if expressed inmammalian cells. The signal peptide for each heavy and light chain areamino acids 1 to 20 of SEQ ID NOs: 2 and 10, and amino acids 1 to 19 ofSEQ ID NOs: 4, 6, and 10.

The nucleotide and amino acid sequences of the light chain of murineSR-1 are set forth in SEQ ID NO: 7 and SEQ ID NO: 8. The nucleotide andamino acid sequences of the heavy chain of murine SR-1 is set forth inSEQ ID NO: 9 and SEQ ID NO: 10. Variants with further substitutions(e.g. conservative substitutions of the murine amino acids) may alsoretain the high binding affinity. Substitutions, deletions or insertionsin positions within the CDRs and framework may be made without affectingaffinity.

In one embodiment, the humanized antibody comprises a light chain thatretains the original murine CDRs of murine SR-1, e.g., positions about44-58, about 74-80 and about 113-121 of SEQ ID NO: 8. In otherembodiments the humanized antibody comprises a heavy chain that retainsthe murine CDRs of murine SR-1, e.g., positions about 50-54, about 68-85and about 118-125 of SEQ ID NO: 10, and has a human derived frameworkregion. As used herein, it is understood that the term “about”contemplates two to five amino acid position changes so long as theaffinity to c-Kit is maintained.

In one embodiment, such agents may comprise an amino acid sequence setforth in SEQ ID NO: 2, 4, or 6. In another embodiment, any of theaforementioned agents comprise an amino acid sequence that is at least80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acidsequence set forth in SEQ ID NO: 2, 4, or 6. In such embodiments, thesequence variation relative to SEQ ID NO: 2, 4, or 6, respectively, mayrepresent, for example, a conservative substitution of the correspondingframework region of an IgG using an alternative human amino acid at thatposition.

In one embodiment, the aforementioned antibodies exhibit an aviditycharacterized by a k_(d) of lower than 10⁻², or lower than 10⁻³, or10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, as determined by surface plasmonresonance (BIAcore analysis). In another embodiment the aforementionedantibodies exhibit a neutral antagonist potency IC50 lower than 1×10⁻²,or lower than 1×10⁻³, or 1×10⁻⁴, 1×10⁻⁵, 1×10⁻⁶, 1×10⁻⁷, 1×10⁻⁸, 1×10⁻⁹,as determined by cellular assays.

The present invention provides a variety of specific binding and neutralantagonist agents, including but not limited to human or humanized c-Kitspecific antibodies, that are derived from murine SR-1 and retaindesirable characteristics such as Kd (dissociation rate constant) forc-Kit in the range of 1×10⁻² or lower, or ranging down to 1×10⁻⁹ orlower, (e.g., 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ or lower)and/or neutral antagonist IC50 for c-Kit in the range of 1×10⁻² orlower, or ranging down to 1×10⁻⁹ or lower, (e.g., 10⁻², 10⁻³, 10⁻⁴,10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ or lower) and/or the ability to reducesymptoms of a c-Kit associated disorder. The invention also providesnucleic acids encoding such specific binding agent polypeptides, vectorsand recombinant host cells comprising such nucleic acids, methods ofproducing such specific binding agents, pharmaceutical formulationsincluding such specific binding agents, methods of preparing thepharmaceutical formulations, and methods of treating patients with thepharmaceutical formulations and compounds.

Nucleic acids encoding these modified light chain variable regions wereconstructed and co-expressed with nucleic acids encoding a CDR-graftedor a humanized heavy chain and vice versa, and optionally may be linkedto constant regions. Any humanized or chimeric heavy chain and lightchains may be combined as long as suitable binding affinity ismaintained. The desired genes were introduced into mammalian cells andthe resultant recombinant immunoglobulin products were expressed,purified and characterized.

The term “antibody” is used in the broadest sense and includes fullyassembled antibodies, monoclonal antibodies (including human, humanizedor chimeric antibodies), polyclonal antibodies, multispecific antibodies(e.g., bispecific antibodies), and antibody fragments that can bindantigen (e.g., Fab′, F′(ab)₂, Fv, single chain antibodies, diabodies),comprising complementarity determining regions (CDRs) of the foregoingas long as they exhibit the desired biological activity. The term“antibody” explicitly excludes murine antibody (i.e. antibody producedby a murine hybridoma or having the same sequence as an antibodyproduced by a murine hybridoma) from the scope of the term.

The term “specific binding agent” includes antibodies as defined aboveand recombinant peptides or other compounds that contain sequencesderived from CDRs having the desired antigen-binding properties.Specifically included in the term are peptides containing amino acidsequences that are at least 80%, 90% or 100% identical to one or moreCDRs of murine SR-1, preferably including heavy chain CDR3.

As used herein, the term “neutral antagonist” is understood to mean aspecific binding agent that is capable of inhibiting an agonistsactivity These agents includes antibodies as defined above andrecombinant peptides or other compounds that contain sequences derivedfrom CDRs having the desired antigen-binding properties. Specificallyincluded in the term are peptides containing amino acid sequences thatare at least 80%, 90% or 100% identical to one or more CDRs of murineSR-1, preferably including heavy chain CDR3.

Also included in the term are “peptibodies” which are moleculescomprising an antibody Fc domain as the “vehicle” attached to at leastone antigen-binding peptide. Antibody CDR's from the SR-1 antibody maybe suitable for incorporation into a peptibody, particularly includingthe CDR3 of the heavy chain. The production of peptibodies is generallydescribed in PCT publication WO 00/24782, published May 4, 2000.Peptides may be linked in tandem (i.e., sequentially), with or withoutlinkers. Peptides containing a cysteinyl residue may be cross-linkedwith another Cys-containing peptide, either or both of which may belinked to a vehicle. Any peptide having more than one Cys residue mayform an intrapeptide disulfide bond, as well. Any of these peptides maybe derivatized, for example the carboxyl terminus may be capped with anamino group, cysteines may be capped, or amino acid residues maysubstituted by moieties other than amino acid residues (see, e.g.,Bhatnagar et al., J. Med. Chem. 39: 3814-9 (1996), and Cuthbertson etal., J. Med. Chem. 40: 2876-82 (1997), which are incorporated byreference herein in their entirety). The peptide sequences may beoptimized, analogous to affinity maturation for antibodies, or otherwisealtered by alanine scanning or random or directed mutagenesis followedby screening to identify the best binders. Lowman, Ann. Rev. Biophys.Biomol. Struct. 26: 401-24 (1997).

Various molecules can be inserted into the specific binding agentstructure, e.g., within the peptide portion itself or between thepeptide and vehicle portions of the specific binding agents, whileretaining the desired activity of specific binding agent. One canreadily insert, for example, molecules such as an Fc domain or fragmentthereof, polyethylene glycol or other related molecules such as dextran,a fatty acid, a lipid, a cholesterol group, a small carbohydrate, apeptide, a cyotoxic agent, a chemotherapeutic agent, a detectable moietyas described herein (including fluorescent agents, radiolabels such asradioisotopes), an oligosaccharide, oligonucleotide, a polynucleotide,interference (or other) RNA, enzymes, hormones, or the like. Othermolecules suitable for insertion in this fashion will be appreciated bythose skilled in the art, and are encompassed within the scope of theinvention. This includes insertion of, for example, a desired moleculein between two consecutive amino acids, optionally joined by a suitablelinker.

An “isolated” antibody is one that has been identified and separatedfrom a component of the cell that expressed it. Contaminant componentsof the cell are materials that would interfere with diagnostic ortherapeutic uses for the antibody, and may include enzymes, hormones,and other proteinaceous or nonproteinaceous solutes. In preferredembodiments, the antibody will be purified (1) to greater than 95% byweight of antibody, and most preferably more than 99% by weight, (2) toa degree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence, or (3) to homogeneity by SDS-PAGE underreducing or nonreducing conditions using Coomassie blue or, preferably,silver stain. Isolated naturally occurring antibody includes theantibody in situ within recombinant cells since at least one componentof the antibody's natural environment will not be present. Ordinarily,however, isolated antibody will be prepared by at least one purificationstep.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst an individual antigenic site or epitope, in contrast topolyclonal antibody preparations that typically include differentantibodies directed against different epitopes.

Depending on the amino acid sequence of the constant domain of theirheavy chains, human immunoglobulins can be assigned to differentclasses. There are five major classes, IgA, IgD, IgE, IgG and IgM, andseveral of these may be further divided into subclasses or isotypes,e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constantdomains that correspond to the different classes of immunoglobulins arecalled alpha, delta, epsilon, gamma and mu respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known. Different isotypes have differenteffector functions; for example, IgG1 and IgG3 isotypes have ADCCactivity.

The term “hypervariable” region refers to the amino acid residues of anantibody which are responsible for antigen-binding. The hypervariableregion comprises amino acid residues from a complementarity determiningregion or CDR [i.e., residues 24-34 (L), 50-56 (L2) and 89-97 (L3) inthe light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102(H3) in the heavy chain variable domain as described by Kabat et al.,Sequences of Proteins of Immunological Interest, 5^(th) Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991)].Even a single CDR may recognize and bind antigen, although with a loweraffinity than the entire antigen binding site containing all of theCDRs. It is understood that the CDR of an antibody may includeadditional or fewer sequences outside the specified limits above so longas the antibody retains it's ability to bind the target molecule.

An alternative definition of residues from a hypervariable “loop” isdescribed by Chothia et al., J. Mol. Biol. 196: 901-917 (1987) asresidues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chainvariable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavychain variable domain.

“Framework” or FR residues are those variable region residues other thanthe hypervariable region residues.

“Antibody fragments” comprise a portion of an intact full lengthantibody, preferably the antigen binding or variable region of theintact antibody. Examples of antibody fragments include Fab, Fab′,F(ab′)2, and Fv fragments; diabodies; linear antibodies (Zapata et al.,Protein Eng., 8(10):1057-1062 (1995)); single-chain antibody molecules;and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment which contains the constant region.The Fab fragment contains all of the variable domain, as well as theconstant domain of the light chain and the first constant domain(C_(H)1) of the heavy chain. The Fc fragment displays carbohydrates andis responsible for many antibody effector functions (such as bindingcomplement and cell receptors), that distinguish one class of antibodyfrom another.

Pepsin treatment yields an F(ab′)2 fragment that has two “Single-chainFv” or “sFv” antibody fragments comprising the V_(H) and V_(L) domainsof antibody, wherein these domains are present in a single polypeptidechain. Fab fragments differ from Fab′ fragments by the inclusion of afew additional residues at the carboxy terminus of the heavy chainC_(H)1 domain including one or more cysteines from the antibody hingeregion. Preferably, the Fv polypeptide further comprises a polypeptidelinker between the V_(H) and V_(L) domains that enables the Fv to formthe desired structure for antigen binding. For a review of sFv seePluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

“Fv” is the minimum antibody fragment that contains a complete antigenrecognition and binding site. This region consists of a dimer of oneheavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen binding site on thesurface of the V_(H) V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, a single variabledomain (or half of an Fv comprising only three CDRs specific for anantigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H) V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and 30 Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies, but can also be produced directly byrecombinant host cells. See, for example, Better et al., Science 240:1041-1043 (1988); Skerra et al. Science 240: 1038-1041 (1988); Carter etal., Bio/Technology 10:163-167 (1992).

As provided herein, the compositions and methods of treatinginflammatory, autoimmune, oncologic and fibrotic disorders may utilizeone or more anti-c-Kit therapeutics used singularly or in combinationwith other therapeutics to achieve the desired effects. Exemplaryanti-fibrotic agents suitable for use in accordance with the inventioninclude cytokines wherein the cytokine is selected from transforminggrowth factor ρ (TGF-β), interleukin-4 (IL-4), interleukin-5 (IL-5),interleukin-9 (IL-9), interleukin-13(IL-13),granulocyte/macrophage-colony stimulating factor (GM-CSF), tumornecrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), connectivetissue growth factor (CTGF), interleukin-6 (IL-6), oncostatin M (OSM),platelet derived growth factor (PDGF), monocyte chemotactic protein 1(CCL2/MCP-1), and pulmonary and activation-regulated chemokine(CCL18/PARC).

Antibodies derived from SR-1 according to the present invention arepreferably produced by recombinant DNA methodology using one of theantibody expression systems well known in the art (see, e.g., Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory(1988)). Such antibodies are also preferably chimeric fusion proteinshaving immunoglobulin derived variable sequences and human constantregions, or more preferably are more human-like monoclonal antibodies(such as human or humanized antibodies) that comprise human antibodyresidues but preferably retain at least the CDRs of murine SR-1. Inaddition to intact, full-length molecules, the term “antibody” alsorefers to fragments thereof or multimers or aggregates of intactmolecules and/or fragments that bind to c-Kit.

The phrase “humanized antibody” refers to an antibody derived from anon-human antibody, typically a mouse monoclonal antibody.Alternatively, a humanized antibody may be derived from a chimericantibody that retains or substantially retains the antigen bindingproperties of the parental, non-human, antibody but which exhibitsdiminished immunogenicity as compared to the parental antibody whenadministered to humans. The phrase “chimeric antibody,” as used herein,refers to an antibody containing sequence derived from two differentantibodies (see, e.g., U.S. Pat. No. 4,816,567) which typicallyoriginate from different species. Most typically, chimeric antibodiescomprise human and murine antibody fragments, generally human constantand mouse variable regions.

Recombinant Production of Antibodies

The amino acid sequence of an immunoglobulin of interest may bedetermined by direct protein sequencing, and suitable encodingnucleotide sequences can be designed according to a universal codontable.

Alternatively, DNA encoding the monoclonal antibodies may be isolatedand sequenced from the hybridoma cells using conventional procedures(e.g., by using oligonucleotide probes that are capable of bindingspecifically to genes encoding the heavy and light chains of themonoclonal antibodies). Sequence determination will generally requireisolation of at least a portion of the gene or cDNA of interest. Usuallythis requires cloning the DNA or, preferably, mRNA (i.e., cDNA) encodingthe monoclonal antibodies. Cloning is carried out using standardtechniques (see, e.g., Sambrook et al. (1989) Molecular Cloning: ALaboratory Guide, Vols 1-3, Cold Spring Harbor Press, which isincorporated herein by reference). For example, a cDNA library may beconstructed by reverse transcription of polyA+ mRNA, preferablymembrane-associated mRNA, and the library screened using probes specificfor human immunoglobulin polypeptide gene sequences. In a preferredembodiment, however, the polymerase chain reaction (PCR) is used toamplify cDNAs (or portions of full-length cDNAs) encoding animmunoglobulin gene segment of interest (e.g., a light chain variablesegment). The amplified sequences can be readily cloned into anysuitable vector, e.g., expression vectors, minigene vectors, or phagedisplay vectors. It will be appreciated that the particular method ofcloning used is not critical, so long as it is possible to determine thesequence of some portion of the immunoglobulin polypeptide of interest.

As used herein, an “isolated” nucleic acid molecule or “isolated”nucleic acid sequence is a nucleic acid molecule that is either (1)identified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe nucleic acid or (2) cloned, amplified, tagged, or otherwisedistinguished from background nucleic acids such that the sequence ofthe nucleic acid of interest can be determined. An isolated nucleic acidmolecule is other than in the form or setting in which it is found innature. However, an isolated nucleic acid molecule includes a nucleicacid molecule contained in cells that ordinarily express the antibodywhere, for example, the nucleic acid molecule is in a chromosomallocation different from that of natural cells.

One source for RNA used for cloning and sequencing is a hybridomaproduced by obtaining a B cell from the transgenic mouse and fusing theB cell to an immortal cell. An advantage of using hybridomas is thatthey can be easily screened, and a hybridoma that produces a humanmonoclonal antibody of interest selected. Alternatively, RNA can beisolated from B cells (or whole spleen) of the immunized animal. Whensources other than hybridomas are used, it may be desirable to screenfor sequences encoding immunoglobulins or immunoglobulin polypeptideswith specific binding characteristics. One method for such screening isthe use of phage display technology. Phage display is described in e.g.,Dower et al., WO 91/17271, McCafferty et al., WO 92/01047, and Caton andKoprowski, Proc. Natl. Acad. Sci. USA, 87:6450-6454 (1990), each ofwhich is incorporated herein by reference. In one embodiment using phagedisplay technology, cDNA from an immunized transgenic mouse (e.g., totalspleen cDNA) is isolated, the polymerase chain reaction is used toamplify a cDNA sequences that encode a portion of an immunoglobulinpolypeptide, e.g., CDR regions, and the amplified sequences are insertedinto a phage vector. cDNAs encoding peptides of interest, e.g., variableregion peptides with desired binding characteristics, are identified bystandard techniques such as panning.

The sequence of the amplified or cloned nucleic acid is then determined.Typically the sequence encoding an entire variable region of theimmunoglobulin polypeptide is determined, however, it will sometimes beadequate to sequence only a portion of a variable region, for example,the CDR-encoding portion. Typically the portion sequenced will be atleast 30 bases in length, more often bases coding for at least aboutone-third or at least about one-half of the length of the variableregion will be sequenced.

Sequencing can be carried out on clones isolated from a cDNA library,or, when PCR is used, after subcloning the amplified sequence or bydirect PCR sequencing of the amplified segment. Sequencing is carriedout using standard techniques (see, e.g., Sambrook et al. (1989)Molecular Cloning: A Laboratory Guide, Vols 1-3, Cold Spring HarborPress, and Sanger, F. et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463-5467, which is incorporated herein by reference). By comparing thesequence of the cloned nucleic acid with published sequences of humanimmunoglobulin genes and cDNAs, one of skill will readily be able todetermine, depending on the region sequenced, (i) the germline segmentusage of the hybridoma immunoglobulin polypeptide (including the isotypeof the heavy chain) and (ii) the sequence of the heavy and light chainvariable regions, including sequences resulting from N-region additionand the process of somatic mutation. One source of immunoglobulin genesequence information is the National Center for BiotechnologyInformation, National Library of Medicine, National Institutes ofHealth, Bethesda, Md.

Once isolated, the DNA may be operably linked to expression controlsequences or placed into expression vectors, which are then transfectedinto host cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to direct the synthesis of monoclonal antibodiesin the recombinant host cells. Recombinant production of antibodies iswell known in the art.

Expression control sequences refer to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is operably linked when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, operably linkedmeans that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

Cell, cell line, and cell culture are often used interchangeably and allsuch designations herein include progeny. Transformants and transformedcells include the primary subject cell and cultures derived therefromwithout regard for the number of transfers. It is also understood thatall progeny may not be precisely identical in DNA content, due todeliberate or inadvertent mutations. Mutant progeny that have the samefunction or biological activity as screened for in the originallytransformed cell are included.

The invention also provides isolated nucleic acids encoding specificbinding agents or antibodies of the invention, optionally operablylinked to control sequences recognized by a host cell, vectors and hostcells comprising the nucleic acids, and recombinant techniques for theproduction of the specific binding agents or antibodies, which maycomprise culturing the host cell so that the nucleic acid is expressedand, optionally, recovering the specific binding agent or antibody fromthe host cell culture or culture medium.

Many vectors are known in the art. Vector components may include one ormore of the following: a signal sequence (that may, for example, directsecretion of the specific binding agent or antibody), an origin ofreplication, one or more selective marker genes (that may, for example,confer antibiotic or other drug resistance, complement auxotrophicdeficiencies, or supply critical nutrients not available in the media),an enhancer element, a promoter, and a transcription terminationsequence, all of which are well known in the art.

Suitable host cells include prokaryote, yeast, or higher eukaryote cellsdescribed above. Suitable prokaryotes for this purpose includeeubacteria, such as Gram-negative or Gram-positive organisms, forexample, Enterobacteriaceae such as Escherichia, e.g., E. coli,Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonellatyphimurium, Serratia, e.g., Serratia marcescans, and Shigella, as wellas Bacilli such as B. subtilis and B. licheniformis, Pseudomonas, andStreptomyces. In addition to prokaryotes, eukaryotic microbes such asfilamentous fungi or yeast are suitable cloning or expression hosts forspecific binding agent-encoding vectors. Saccharomyces cerevisiae, orcommon baker's yeast, is the most commonly used among lower eukaryotichost microorganisms. However, a number of other genera, species, andstrains are commonly available and useful herein, such as Pichia, e.g.P. pastoris, Schizosaccharomyces pombe; Kluyveromyces, Yarrowia;Candida; Trichoderma reesia; Neurospora crassa; Schwanniomyces such asSchwanniomyces occidentalis; and filamentous fungi such as, e.g.,Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A.nidulans and A. niger.

Suitable host cells for the expression of glycosylated specific bindingagent or antibody are derived from multicellular organisms. Examples ofinvertebrate cells include plant and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection of such cells are publicly available, e.g., theL-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyxmori NPV.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become routineprocedure. Examples of useful mammalian host cell lines are Chinesehamster ovary cells, including CHOK1 cells (ATCC CCL61), DXB-11, DG-44,and Chinese hamster ovary cells/−DHFR (CHO, Urlaub et al., Proc. Natl.Acad. Sci. USA 77: 4216 (1980)); monkey kidney CV1 line transformed bySV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293cells subcloned for growth in suspension culture, [Graham et al., J. GenVirol. 36: 59 (1977)]; baby hamster kidney cells (BHK, ATCC CCL 10);mouse sertoli cells (TM4, Mather, Biol. Reprod. 23: 243-251 (1980));monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells(VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells(BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); humanhepatoma cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCCCCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383: 44-68(1982)); MRC 5 cells or FS4 cells.

Host cells are transformed or transfected with the above-describednucleic acids or vectors for specific binding agent or antibodyproduction and cultured in conventional nutrient media modified asappropriate for inducing promoters, selecting transformants, oramplifying the genes encoding the desired sequences. In addition, novelvectors and transfected cell lines with multiple copies of transcriptionunits separated by a selective marker are particularly useful andpreferred for the expression of specific binding agents or antibodies.

The host cells used to produce the specific binding agent or antibody ofthis invention may be cultured in a variety of media. Commerciallyavailable media such as Ham's F10 (Sigma), Minimal Essential Medium((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle'sMedium ((DMEM), Sigma) are suitable for culturing the host cells. Inaddition, any of the media described in Ham et al., Meth. Enz. 58: 44(1979), Barnes et al., Anal. Biochem. 102: 255 (1980), U.S. Pat. Nos.4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90103430; WO87/00195; or U.S. Pat. Re. No. 30,985 may be used as culture media forthe host cells. Any of these media may be supplemented as necessary withhormones and/or other growth factors (such as insulin, transferrin, orepidermal growth factor), salts (such as sodium chloride, calcium,magnesium, and phosphate), buffers (such as HEPES), nucleotides (such asadenosine and thymidine), antibiotics (such as Gentamycin™ drug), traceelements (defined as inorganic compounds usually present at finalconcentrations in the micromolar range), and glucose or an equivalentenergy source. Any other necessary supplements may also be included atappropriate concentrations that would be known to those skilled in theart. The culture conditions, such as temperature, pH, and the like, arethose previously used with the host cell selected for expression, andwill be apparent to the ordinarily skilled artisan. The expressionvectors, pDC323 and pDC324 as described in U.S. Patent Application No.20030082735, containing the appropriate respective light chain and heavychain pair were transfected into the CS9 host cell line.

Upon culturing the host cells, the specific binding agent or antibodycan be produced intracellularly, in the periplasmic space, or directlysecreted into the medium. If the specific binding agent or antibody isproduced intracellularly, as a first step, the particulate debris,either host cells or lysed fragments, is removed, for example, bycentrifugation or ultrafiltration.

The specific binding agent or antibody composition can be purifiedusing, for example, hydroxylapatite chromatography, cation or anionexchange chromatography, or preferably affinity chromatography, usingthe antigen of interest or protein A or protein G as an affinity ligand.Protein A can be used to purify specific binding agents or antibodiesthat are based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J.Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended for all mouseisotypes and for human γ3 (Guss et al., EMBO J. 5: 15671575 (1986)). Thematrix to which the affinity ligand is attached is most often agarose,but other matrices are available. Mechanically stable matrices such ascontrolled pore glass or poly(styrenedivinyl)benzene allow for fasterflow rates and shorter processing times than can be achieved withagarose. Where the specific binding agent or antibody comprises a C_(H)3 domain, the Bakerbond ABX™ resin (J. T. Baker, Phillipsburg, N.J.) isuseful for purification. Other techniques for protein purification suchas ethanol precipitation, Reverse Phase HPLC, chromatofocusing,SDS-PAGE, and ammonium sulfate precipitation are also possible dependingon the specific binding agent or antibody to be recovered.

Chimeric and Humanized Antibodies

Chimeric monoclonal antibodies, in which the variable Ig domains of arodent monoclonal antibody are fused to human constant Ig domains, canbe generated using standard procedures known in the art (See Morrison,S. L., et al. (1984) Chimeric Human Antibody Molecules; Mouse AntigenBinding Domains with Human Constant Region Domains, Proc. Natl. Acad.Sci. USA 81, 6841-6855; and, Boulianne, G. L., et al, Nature 312,643-646. (1984)). Although some chimeric monoclonal antibodies haveproved less immunogenic in humans, the rodent variable Ig domains canstill lead to a significant human anti-rodent response.

Humanized antibodies may be achieved by a variety of methods including,for example: (1) grafting the non-human complementarity determiningregions (CDRs) onto a human framework and constant region (a processreferred to in the art as humanizing through “CDR grafting”), or,alternatively, (2) transplanting the entire non-human variable domains,but “cloaking” them with a human-like surface by replacement of surfaceresidues (a process referred to in the art as “veneering”). Thesemethods are disclosed in, e.g., Jones et al., Nature 321:522 525 (1986);Morrison et al., Proc. Natl. Acad. Sci., U.S.A., 81:6851 6855 (1984);Morrison and Oi, Adv. Immunol., 44:65 92 (1988); Verhoeyer et al.,Science 239:1534 1536 (1988); Padlan, Molec. Immun. 28:489 498 (1991);Padlan, Molec. Immunol. 31(3):169 217 (1994); and Kettleborough, C. A.et al., Protein Eng. 4(7):773 83 (1991) each of which is incorporatedherein by reference.

In particular, a rodent antibody on repeated in vivo administration inman either alone or as a conjugate will bring about an immune responsein the recipient against the rodent antibody; the so-called HAMAresponse (Human Anti Mouse Antibody). The HAMA response may limit theeffectiveness of the pharmaceutical if repeated dosing is required. Theimmunogenicity of the antibody may be reduced by chemical modificationof the antibody with a hydrophilic polymer such as polyethylene glycolor by using the methods of genetic engineering to make the antibodybinding structure more human like.

CDR grafting involves introducing one or more of the six CDRs from themouse heavy and light chain variable Ig domains into the appropriateframework regions of a human variable Ig domain. This technique(Riechmann, L., et al., Nature 332, 323 (1988)), utilizes the conservedframework regions (FR1-FR4) as a scaffold to support the CDR loops whichare the primary contacts with antigen. A significant disadvantage of CDRgrafting, however, is that it can result in a humanized antibody thathas a substantially lower binding affinity than the original mouseantibody, because amino acids of the framework regions can contribute toantigen binding, and because amino acids of the CDR loops can influencethe association of the two variable Ig domains. Chimeric SR-1 antibodiesdid not show appropriate functional potency in cell based assays.

To maintain the affinity of the humanized monoclonal antibody, the CDRgrafting technique can be improved by choosing human framework regionsthat most closely resemble the framework regions of the original mouseantibody, and by site-directed mutagenesis of single amino acids withinthe framework or CDRs aided by computer modeling of the antigen bindingsite (e.g., Co, M. S., et al. (1994), J. Immunol. 152, 2968-2976).

One method of humanizing antibodies comprises aligning the non-humanheavy and light chain sequences to human heavy and light chainsequences, selecting and replacing the non-human framework with a humanframework based on such alignment, molecular modeling to predict theconformation of the humanized sequence and comparing to the conformationof the parent antibody. This process is followed by repeated backmutation of residues in the CDR region which disturb the structure ofthe CDRs until the predicted conformation of the humanized sequencemodel closely approximates the conformation of the non-human CDRs of theparent non-human antibody. Such humanized antibodies may be furtherderivatized to facilitate uptake and clearance, e.g., via Ashwellreceptors (See, e.g., U.S. Pat. Nos. 5,530,101 and 5,585,089).

A number of humanizations of mouse monoclonal antibodies by rationaldesign have been reported (See, for example, 20020091240 published Jul.11, 2002, WO 92/11018 and U.S. Pat. No. 5,693,762, U.S. Pat. No.5,766,866. Human engineering of antibodies have also been described in,e.g., Studnicka et al. U.S. Pat. No. 5,766,886; Studnicka et al. ProteinEngineering 7: 805-814 (1994).

Production of Antibody Variants

Amino acid sequence variants of the desired specific binding agent orantibody may be prepared by introducing appropriate nucleotide changesinto the encoding DNA, or by peptide synthesis. Such variants include,for example, deletions and/or insertions and/or substitutions ofresidues within the amino acid sequences of the specific binding agentsor antibodies. Any combination of deletion, insertion, and substitutionis made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics. The amino acid changesalso may alter post-translational processes of the specific bindingagent or humanized or variant antibody, such as changing the number orposition of glycosylation sites.

Nucleic acid molecules encoding amino acid sequence variants of thespecific binding agent or antibody are prepared by a variety of methodsknown in the art. Such methods include oligonucleotide-mediated (orsite-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis ofan earlier prepared variant or a non-variant version of the specificbinding agent or antibody.

A useful method for identification of certain residues or regions of thespecific binding agent or antibody that are preferred locations formutagenesis is called “alanine scanning mutagenesis,” as described byCunningham and Wells Science, 244:1081-1085 (1989). Here, a residue orgroup of target residues are identified (e.g., charged residues such asarg, asp, his, lys, and glu) and replaced by a neutral or negativelycharged amino acid (most preferably alanine or polyalanine) to affectthe interaction of the amino acids with antigen. Those amino acidlocations demonstrating functional sensitivity to the substitutions thenare refined by introducing further or other variants at, or for, thesites of substitution. Thus, while the site for introducing an aminoacid sequence variation is predetermined, the nature of the mutation perse need not be predetermined. For example, to analyze the performance ofa mutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed variants arescreened for the desired activity.

Ordinarily, amino acid sequence variants of the specific binding agentor antibody will have an amino acid sequence having at least 60% aminoacid sequence identity with the original specific binding agent orantibody (murine or humanized) amino acid sequences of either the heavyor the light chain, or at least 65%, or at least 70%, or at least 75% orat least 80% identity, more preferably at least 85% identity, even morepreferably at least 90% identity, and most preferably at least 95%identity, including for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.Identity or homology with respect to this sequence is defined herein asthe percentage of amino acid residues in the candidate sequence that areidentical with the original sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions (as definedin Table I below) as part of the sequence identity. None of N-terminal,C-terminal, or internal extensions, deletions, or insertions into thespecific binding agent or antibody sequence shall be construed asaffecting sequence identity or homology. Thus, sequence identity can bedetermined by standard methods that are commonly used to compare thesimilarity in position of the amino acids of two polypeptides. Using acomputer program such as BLAST or FASTA, two polypeptides are alignedfor optimal matching of their respective amino acids (either along thefull length of one or both sequences, or along a pre-determined portionof one or both sequences). The programs provide a default openingpenalty and a default gap penalty, and a scoring matrix such as PAM 250[a standard scoring matrix; see Dayhoff et al., in Atlas of ProteinSequence and Structure, vol. 5, supp. 3 (1978)] can be used inconjunction with the computer program. For example, the percent identitycan then be calculated as: the total number of identical matchesmultiplied by 100 and then divided by the sum of the length of thelonger sequence within the matched span and the number of gapsintroduced into the longer sequences in order to align the twosequences.

Insertions

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intra-sequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includea specific binding agent or antibody with an N-terminal methionylresidue or the specific binding agent or antibody (including antibodyfragment) fused to an epitope tag or a salvage receptor epitope. Otherinsertional variants of the specific binding agent or antibody moleculeinclude the fusion to a polypeptide which increases the serum half-lifeof the specific binding agent or antibody, e.g. at the N-terminus orC-terminus.

Examples of epitope tags include the flu HA tag polypeptide and itsantibody 12CA5 [Field et al., Mol. Cell. Biol. 8: 2159-2165 (1988)]; thec-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto[Evan et al., Mol. Cell. Biol. 5(12): 3610-3616 (1985)]; and the HerpesSimplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al.,Protein Engineering 3(6): 547-553 (1990)]. Other exemplary tags are apoly-histidine sequence, generally around six histidine residues, thatpermits isolation of a compound so labeled using nickel chelation. Otherlabels and tags, such as the FLAG® tag (Eastman Kodak, Rochester, N.Y.)are well known and routinely used in the art.

The term “salvage receptor binding epitope” refers to an epitope of theFc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, or IgG₄) that isresponsible for increasing the in vivo serum half-life of the IgGmolecule.

Substitutions

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the specific bindingagent or antibody molecule removed and a different residue inserted inits place. Substitutional mutagenesis within any of the hypervariable orCDR regions or framework regions is contemplated. Conservativesubstitutions are shown in Table 1. The most conservative substitutionis found under the heading of “preferred substitutions”. If suchsubstitutions result in no change in biological activity, then moresubstantial changes, denominated “exemplary substitutions” in Table 1,or as further described below in reference to amino acid classes, may beintroduced and the products screened.

Table 1 Original Exemplary Preferred Residue Substitutions

-   -   Ala (A) val; leu; ile val    -   Arg (R) lys; gin; asn lys    -   Asn (N) gin; his; asp, lys; gin arg    -   Asp (D) glu; asn glu    -   Cys (C) ser; ala ser    -   Gln (Q) asn; glu asn    -   Glu (E) asp; gin asp    -   Gly (G) ala    -   His (H) asn; gin; lys; arg    -   Ile (I) leu; val; met; ala; leu        -   phe; norleucine    -   Leu (L) norleucine; ile; val; ile        -   met; ala; phe    -   Lys (K) arg; gin; asn arg    -   Met (M) leu; phe; ile leu    -   Phe (F) leu; val; ile; ala; tyr    -   Pro (P) ala    -   Ser (S) thr    -   Thr (T) ser ser    -   Trp (W) tyr; phe tyr    -   Tyr (Y) trp; phe; thr; ser phe    -   Val (V) ile; leu; met; phe; leu        -   ala; norleucine

Substantial modifications in the biological properties of the specificbinding agent or antibody are accomplished by selecting substitutionsthat differ significantly in their effect on maintaining (a) thestructure of the polypeptide backbone in the area of the substitution,for example, as a sheet or helical conformation, (b) the charge orhydrophobicity of the molecule at the target site, or (c) the bulk ofthe side chain. Naturally occurring residues are divided into groupsbased on common side-chain properties:

-   -   (1) hydrophobic: norleucine, met, ala, val, leu, ile;    -   (2) neutral hydrophilic: cys, ser, thr;    -   (3) acidic: asp, glu;    -   (4) basic: asn, gin, his, lys, arg;    -   (5) residues that influence chain orientation: gly, pro; and    -   (6) aromatic: trp, tyr, phe.

Conservative substitutions involve replacing an amino acid with anothermember of its class. Non-conservative substitutions involve replacing amember of one of these classes with a member of another class.

Any cysteine residue not involved in maintaining the proper conformationof the specific binding agent or humanized or variant antibody also maybe substituted, generally with serine, to improve the oxidativestability of the molecule and prevent aberrant crosslinking. Conversely,cysteine bond(s) may be added to the specific binding agent or antibodyto improve its stability (particularly where the antibody is an antibodyfragment such as an Fv fragment).

Affinity Maturation

Affinity maturation involves preparing and screening specific bindingagent or antibody variants that have mutations (deletions, insertions orsubstitutions) within the CDRs of a parent specific binding agent orantibody and selecting variants that have improved biological propertiessuch as binding affinity relative to the parent specific binding agentor antibody. A convenient way for generating such substitutionalvariants is affinity maturation using phage display. Briefly, severalhypervariable region sites (e.g. 6-7 sites) are mutated to generate allpossible amino substitutions at each site. The specific binding agent orantibody variants thus generated may be displayed in a monovalentfashion from filamentous phage particles as fusions to the gene IIIproduct of M13 packaged within each particle. The phage-displayedvariants are then screened for their biological activity (e.g. bindingaffinity).

Alanine scanning mutagenesis can be performed to identify hypervariableregion residues that contribute significantly to antigen binding.Alternatively, or in addition, it may be beneficial to analyze a crystalstructure of the antigen-antibody complex to identify contact pointsbetween the specific binding agent or antibody and the antigen. Suchcontact residues and neighboring residues are candidates forsubstitution according to the techniques elaborated herein. Once suchvariants are generated, the panel of variants is subjected to screeningas described herein and specific binding agents or antibodies withsuperior properties in one or more relevant assays may be selected forfurther development.

Techniques utilizing gene shuffling and directed evolution may also beused to prepare and screen specific binding agent or antibody variantsfor desired activity. For example, Jermutus et al., Proc Natl Acad SciUSA. 2001 Jan. 2; 98(1):75-80 reports that tailored in vitro selectionstrategies based on ribosome display were combined with in vitrodiversification by DNA shuffling to evolve either the off-rate orthermodynamic stability of single-chain Fv antibody fragments (scFvs);Fermer et al., Tumour Biol. 2004 January-April; 25(1-2):7-13 reportsthat use of phage display in combination with DNA shuffling raisedaffinity by almost three orders of magnitude.

Altered Glycosylation

Specific binding agent or antibody variants can also be produced thathave a modified glycosylation pattern relative to the parent specificbinding agent or antibody, for example, deleting one or morecarbohydrate moieties found in the specific binding agent or antibody,and/or adding one or more glycosylation sites that are not present inthe specific binding agent or antibody.

Glycosylation of polypeptides including antibodies is typically eitherN-linked or O-linked. N-linked refers to the attachment of thecarbohydrate moiety to the side chain of an asparagine residue. Thetripeptide sequences asparagine-X-serine and asparagine-X-threonine,where X is any amino acid except proline, are the recognition sequencesfor enzymatic attachment of the carbohydrate moiety to the asparagineside chain. The presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. Thus, N-linkedglycosylation sites may be added to a specific binding agent or antibodyby altering the amino acid sequence such that it contains one or more ofthese tripeptide sequences. O-linked glycosylation refers to theattachment of one of the sugars N-aceylgalactosamine, galactose, orxylose to a hydroxyamino acid, most commonly serine or threonine,although 5-hydroxyproline or 5-hydroxylysine may also be used. O-linkedglycosylation sites may be added to a specific binding agent or antibodyby inserting or substituting one or more serine or threonine residues tothe sequence of the original specific binding agent or antibody.

Other Modifications

Cysteine residue(s) may be removed or introduced in the Fc region,thereby eliminating or increasing interchain disulfide bond formation inthis region. The homodimeric specific binding agent or antibody thusgenerated may have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med. 176: 1191-1195 (1992)and Shopes, B. J. Immunol. 148: 2918-2922 (1992). Homodimeric specificbinding agents or antibodies may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al., CancerResearch 53: 2560-2565 (1993). Alternatively, a specific binding agentor antibody can be engineered which has dual Fc regions and may therebyhave enhanced complement lysis and ADCC capabilities. See Stevenson etal., Anti-CancerDrug Design 3: 219-230 (1989).

It has been shown that sequences within the CDR can cause an antibody tobind to MHC Class II and trigger an unwanted helper T-cell response. Aconservative substitution can allow the specific binding agent orantibody to retain binding activity yet reduce its ability to trigger anunwanted T-cell response.

It is also contemplated that one or more of the N-terminal 20 aminoacids of the heavy or light chain are removed.

Modifications to increase serum half-life also may desirable, forexample, by incorporation of or addition of a salvage receptor bindingepitope (e.g., by mutation of the appropriate region or by incorporatingthe epitope into a peptide tag that is then fused to the specificbinding agent or antibody at either end or in the middle, e.g., by DNAor peptide synthesis) (see, e.g., WO96/32478) or adding molecules suchas PEG or other water soluble polymers, including polysaccharidepolymers.

The salvage receptor binding epitope preferably constitutes a regionwherein any one or more amino acid residues from one or two loops of aFc domain are transferred to an analogous position of the specificbinding agent or antibody or fragment. Even more preferably, three ormore residues from one or two loops of the Fc domain are transferred.Still more preferred, the epitope is taken from the C_(H)2 domain of theFc region (e.g., of an IgG) and transferred to the C_(H)1, C_(H)3, orV_(H) region, or more than one such region, of the specific bindingagent or antibody. Alternatively, the epitope is taken from the C_(H)2domain of the Fc region and transferred to the C_(L) region or V_(L)region, or both, of the specific binding agent or antibody fragment. Seealso International applications WO 97/34631 and WO 96/32478 whichdescribe Fc variants and their interaction with the salvage receptor.

The regulation of IgG homeostasis in vivo depends upon its binding tothe FcRn. Modification of the interaction between the Fc domain of IgGand FcRn has been reported to improve the serum half-life of monoclonalantibodies. Mutations in the Fc that result in higher affinity bindingto neonatal Fc receptor FcRn and slowing degradation and improving PKprofile would be preferable. The FcRn binding site on IgG is located atthe C_(H)2-C_(H)3 domain interface. Mutations of residues in this area(M428L and T250Q/M428L, T250Q/M428L, P257I/Q311I, M252Y/S254T/T256E,H433K/N434F/Y436H, or M252Y/S254T/T256E/H433K/N434F/Y436H) result inincreased affinity of IgG1 for human FcRn at pH 6.0 and pH 7.3.Additionally, some of these mutations resulted in enhancedpharmacokinetic properties (slower clearance, longer half-life) whengiven intravenously to monkeys.

Other sites of the constant region have been identified that areresponsible for complement dependent cytotoxicity (CDC), such as the C1qbinding site and/or the antibody-dependent cellular cytotoxicity (ADCC)[see, e.g., Molec. Immunol. 29 (5): 633-9 (1992); Shields et al., J.Biol. Chem., 276(9):6591-6604 (2001), incorporated by reference hereinin its entirety]. Mutation of residues within Fc receptor binding sitescan result in altered (i.e. increased or decreased) effector function,such as altered ADCC or CDC activity, or altered half-life. As describedabove, potential mutations include insertion, deletion or substitutionof one or more residues, including substitution with alanine, aconservative substitution, a non-conservative substitution, orreplacement with a corresponding amino acid residue at the same positionfrom a different subclass (e.g. replacing an IgG1 residue with acorresponding IgG2 residue at that position).

Other Covalent Modifications

Covalent modifications of the specific binding agent or antibody arealso included within the scope of this invention. They may be made bychemical synthesis or by enzymatic or chemical cleavage of the specificbinding agent or antibody, if applicable. Other types of covalentmodifications can be introduced into the specific binding agent orantibody by reacting targeted amino acid residues with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,.alpha.-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino-terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing .alpha.-amino-containing residues includeimidoesters such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R—N.dbd.C.dbd.N—R′), where R and R′ aredifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively. Theseresidues are deamidated under neutral or basic conditions. Thedeamidated form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the .alpha.-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86(1983)), acetylation of the N-terminal amine, and amidation of anyC-terminal carboxyl group.

Another type of covalent modification involves chemically orenzymatically coupling glycosides to the specific binding agent orantibody. These procedures are advantageous in that they do not requireproduction of the specific binding agent or antibody in a host cell thathas glycosylation capabilities for N- or O-linked glycosylation.Depending on the coupling mode used, the sugar(s) may be attached to (a)arginine and histidine, (b) free carboxyl groups, (c) free sulfhydrylgroups such as those of cysteine, (d) free hydroxyl groups such as thoseof serine, threonine, or hydroxyproline, (e) aromatic residues such asthose of phenylalanine, tyrosine, or tryptophan, or (f) the amide groupof glutamine. These methods are described in WO87/05330 published 11Sep. 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp.259-306 (1981).

Removal of any carbohydrate moieties present on the specific bindingagent or antibody may be accomplished chemically or enzymatically.Chemical deglycosylation requires exposure of the specific binding agentor antibody to the compound trifluoromethanesulfonic acid, or anequivalent compound. This treatment results in the cleavage of most orall sugars except the linking sugar (N-acetylglucosamine orN-acetylgalactosamine), while leaving the specific binding agent orantibody intact. Chemical deglycosylation is described by Hakimuddin, etal. Arch. Biochem. Biophys. 259: 52 (1987) and by Edge et al. Anal.Biochem., 118: 131 (1981). Enzymatic cleavage of carbohydrate moietieson a specific binding agent or antibody can be achieved by the use of avariety of endo- and exo-glycosidases as described by Thotakura et al.Meth. Enzymol. 138: 350 (1987).

Another type of covalent modification of the specific binding agent orantibody comprises linking the specific binding agent or antibody to oneof a variety of nonproteinaceous polymers, e.g., polyethylene glycol,polypropylene glycol, polyoxyethylated polyols, polyoxyethylatedsorbitol, polyoxyethylated glucose, polyoxyethylated glycerol,polyoxyalkylenes, or polysaccharide polymers such as dextran. Suchmethods are known in the art, see, e.g. U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192, 4,179,337, 4,766,106,4,179,337, 4,495,285, 4,609,546 or EP 315 456.

Therapeutic Uses

“Treatment” is an intervention performed with the intention ofpreventing the development or altering the pathology of a disorder.Accordingly, “treatment” refers to both therapeutic treatment andprophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those in which thedisorder is to be prevented.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal is human.

As used herein, the phrase “therapeutically effective amount” is meantto refer to an amount of therapeutic or prophylactic humanized c-Kitantibody that provides a reduction in mast cell or progenitor cellnumber and/or activity, reduction in fibroid elements or theirprecursors, or that provides a reduction in the severity or progressionof symptoms associated with c-kit associated disease (i.e. that provides“therapeutic efficacy”). Mast cells and progenitor hematopoieticpluripotent stem cells are the primary cell types expressing c-Kit andthus it is contemplated that cells derived from HSC such as mast cellsand that are involved in diseases can be treated with the compositionsand methods of the invention.

The phrase “fibrotic-reducing activity” is meant to refer to the abilityto inhibit, fully or partially and reverse inflammation resulting fromimmune system activation and fibrosis.

As used herein the term “fibrotic disease or disorder” refers toconditions involving fibrosis in one or more tissues. As used herein theterm “fibrosis” refers to abberant formation or development of excessfibrous connective tissue in an organ or tissue as a reactive process,as opposed to formation of fibrous tissue as a normal constituent orhealing of an organ or tissue. Fibrosis is characterized by fibroblastaccumulation and collagen deposition in excess of normal deposition inany particular tissue. As used herein the term “fibrosis” is usedsynonymously with “aberrant healing, involving mesenchymal-fibroblastcell transformation, excessive fibroblast proliferation, activity anddeposition of collagens and other extracellular matrix proteins”.

Fibroblasts are connective tissue cells, which are dispersed inconnective tissue throughout the body. Fibroblasts secrete a nonrigidextracellular matrix containing type I and/or type III collagen. Inresponse to an injury to a tissue, nearby fibroblasts or mesenchymalprecursor cells in circulation migrate into the wound, may becomealternatively activated under the influence of other cells such as mastcells and their mediators, proliferate, and produce large amounts ofcollagenous extracellular matrix. Collagen is a fibrous protein rich inglycine and proline that is a major component of the extracellularmatrix and connective tissue, cartilage, and bone. Collagen moleculesare triple-stranded helical structures called α-chains, which are woundaround each other in a ropelike helix. Collagen exists in several formsor types; of these, type I, the most common, is found in skin, tendon,and bone; and type III is found in skin, blood vessels, and internalorgans.

Mast cell associated fibrotic diseases include pathological fibrosis orscarring (including endocardial sclerosis), idiopathic interstitialfibrosis, interstitial pulmonary fibrosis, perimuscular fibrosis,Symmers' fibrosis, pericentral fibrosis, hepatitis, dermatofibroma,billary cirrhosis, alcoholic cirrhosis, acute pulmonary fibrosis,idiopathic pulmonary fibrosis, acute respiratory distress syndrome,kidney fibrosis/glomerulonephritis, kidney fibrosis/diabeticnephropathy, scleroderma/systemic, scleroderma/local, keloids,hypertrophic scars, severe joint adhesions/arthritis, myelofibrosis,corneal scarring, cystic fibrosis, muscular dystrophy (duchenne's),cardiac fibrosis, muscular fibrosis/retinal separation, esophagealstricture and payronles disease. Further fibrotic disorders may beinduced or initiated by surgery, including scar revision/plasticsurgeries, glaucoma, cataract fibrosis, corneal scarring, jointadhesions, graft vs. host disease, tendon surgery, nerve entrapment,dupuytren's contracture, OB/GYN adhesions/fibrosis, pelvic adhesions,peridural fibrosis, restenosis. It is also contemplated that fibroticconditions where deposition of fibronectin is a causative factor can betreated according to the invention. Idiopathic pulmonary fibrosis,bleomycin lung, cystic fibrosis, and glomerular nephropathy, includingdisease characterized by Fn deposits in the kidneys ultimately leadingto renal failure are examples of conditions which can also be treated inaccordance with the present invention. Inflammation involving activationof the immune system and where mast cells secrete inflammatory cytokinessuch as TNF, and can activate and directly interact with lymphocytes canalso be treated in accordance with the present invention.

Scleroderma is believed to be an autoimmune disease of the connectivetissue resulting in a fibrotic disorder characterized by a thickeningand induration of the skin caused by the overproduction of new collagenby fibroblasts in skin and other organs. Scleroderma may occur as alocal or systemic disease involving a number of organs. Scleroderma isalso referred to as systemic sclerosis. The development of sclerodermapathologies is associated with increased mast cell numbers in theaffected disease tissues/organs.

Systemic sclerosis is characterized by formation of hyalinized andthickened collagenous fibrous tissue, with thickening of the skin andadhesion to underlying tissues, especially of the hands and face. Thedisease may also be characterized by dysphagia due to loss ofperistalsis and submucosal fibrosis of the esophagus, dyspnea due topulmonary fibrosis, myocardial fibrosis, and renal vascular changes.(Stedman's Medical Dictionary, 26^(th) Edition, Williams & Wilkins,1995)). Pulmonary fibrosis affects 30 to 70% of scleroderma patients,often resulting in restrictive lung disease (Atamas et al. Cytokine andGrowth Factor Rev 14: 537-550 (2003)). Some patients have an overlap ofscleroderma and other connective tissue diseases, such as rheumatoidarthritis, systemic lupus erythematosus, and polymyositis. When featuresof scleroderma are present along with features of polymyositis andsystemic lupus erythematosus, the condition is referred to as mixedconnective tissue disease (MCTD).

It is known that the symptoms present in some forms of dermatitis arecaused by degranulation of cutaneous mast cells, resulting in, interalia, histamine release. Thus, another mast cell associated disordersuitable for treatment according to the invention is urticariapigmentosa. This disorder presents characteristic skin lesions that aresingle or multiple pigmented macules or nodules that urticate on rubbingand contain large numbers of mast cells. There are different forms ofassociated dermatitis (inflammation of skin) such as erythema, edema,papular eruptions and pruritus may be present in both human and animaldermatitides, all of which are treatable according to the invention.

Mastocytosis is in many cases a neoplastic disease and involves new orabnormal mast cell growth and may be a consequence of elevated SCFautocrine signaling or an activating c-Kit mutation. Mastocytosis may belimited or systemic involving multiple organs such as the bone marrow.Mast cells release certain mediators, or chemicals, of which one ishistamine, into the body in response to certain events. People withsystemic mastocytosis develop an increase in the number of mast cells,or they develop abnormally shaped mast cells, which may not functionproperly. In addition, the mast cells fail to die off when they aresupposed to, further increasing the total mast cell burden. When themast cells degranulate and release their contents it can cause manyacute and potentially serious conditions or diseases. Mast celldisorders also include proliferative disorders resulting in localizeddisease such as solitary cutaneous mastocytoma up to the more severedisease of mast cell leukemia. Examples include cutaneous mastocytoma,aggressive mastocytosis, indolent mastocytosis, mastocytosis withassociated hematologic disorder, urticaria pigmentosa, telangiectasiamacularis eruptiva perstans (tmep), systemic mast cell disease, mastcell leukemia, myeloid leukemia, systemic mastocytosis (with or withoutcutaneous manifestations such as urticaria pigmentosa), mast cellactivation syndrome/disorder, and more common pediatric mast celldisorders such as solitary mastocytoma and diffuse cutaneousmastocytosis.

Mast cell activation syndrome or disorder is characterized by a normalor nearly normal number of mast cells. However, the mast cells areeasily triggered to release their contents, which results in many of thesame symptoms. The danger of anaphylaxis and shock is present with thisdisorder, but unlike proliferative disorders of mast cells, thissyndrome may not have the potential to progress to a more aggressive ormalignant stage. Examples of such disorders associated with mast celldegranulation can include abdominal pain, hives and rashes, anaphylaxis,inflammation of the esophagus, blood pressure changes and shock,intestinal cramping and bloating, bone pain (mild tosevere/debilitating), itching, with and without rashes, chest pain,liver, spleen and other organ involvement, cognitive difficulties/brainfog, malabsorption, degenerative disc disease, migraine headaches,diarrhea, muscle pain, dizziness/vertigo/lightheadedness, nausea,faintness, osteoporosis/osteopenia, fatigue, peripheral neuropathy andparesthesias, flushing, rapid heart rate, gastroesophageal reflux, andvomiting.

The role of mast cells in allergic diseases has been clinicallyvalidated by drugs that block mast cell specific mediators such ashistamine and corticosteroids which among their activities cause mastcell apoptosis. Additional mast cell related diseases includehistamine-mediated allergic reactions that can be treated by inhibitingchemokine-induced mast cell and basophil degranulation and release ofhistamine. Examples of mast cell associated disorders or diseases whichmay be effectively treated with the subject methods and compositionsalso include, but are not limited to, contact dermatitis, atopicdermatitis, allergic dermatitis, eczematous dermatitis and dermatitiscaused by insect bites or stings.

Other mast cell related indications suitable for treatment by themethods and compositions of the invention include pulmonary inflammatoryconditions in interstitial lung diseases for example sarcoidosis,neonatal respiratory distress syndrome (RDS), bronchopulmonary dysplasia(BPD), and conditions characterized by an elevation in serum PLA2activity, such as adult RDS (ARDS).

Mast cells have also been published to show roles in arthritis. Mastcells are increased in the inflamed synovial tissues of RA and OApatients, and Gleevec has been shown to cause mast cell apoptosis insynovial explants and human case studies show efficacy in RA patients.Mast cells have roles in septic shock, pancreatitis, collagen vasculardiseases, acute renal failure, peritonitis, and autoimmune uveitis.

Studies also suggest that mast cells participate in the pathophysiologyof multiple sclerosis. It is thought that mast cells in the brainrelease vasoactive amines which may cause demyelination. Histaminereleased from mast cells may alter blood vessel integrity and causepartial breakdown of the blood-brain barrier again implicated in theetiology of multiple sclerosis. Thus, it is contemplated that themethods and compositions of the invention are suitable for treatment oramelioration of the morbidity associated with multiple sclerosis.

C-kit is also expressed on certain non-immune cells such as melanocytesand intestinal cells as well as spermatocytes. The invention may haveutility in the treatment of melanoma and GIST and may have utility as amale contraceptive.

Administration and Preparation of Pharmaceutical Formulations

The anti-c-Kit specific binding agents or antibodies used in thepractice of a method of the invention may be formulated intopharmaceutical compositions comprising a carrier suitable for thedesired delivery method. Suitable carriers include any material which,when combined with the anti-c-Kit specific binding and neutralantagonist agent or antibody, retains the high-affinity binding andpotency at c-Kit and is nonreactive with the subject's immune systems.Examples include, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like. A variety of aqueouscarriers may be used, e.g., water, buffered water, 0.4% saline, 0.3%glycine and the like, and may include other proteins for enhancedstability, such as albumin, lipoprotein, globulin, etc., subjected tomild chemical modifications or the like.

Exemplary antibody concentrations in the formulation may range fromabout 0.1 mg/ml to about 180 mg/ml or from about 0.1 mg/mL to about 50mg/mL, or from about 0.5 mg/mL to about 25 mg/mL, or alternatively fromabout 2 mg/mL to about 10 mg/mL. An aqueous formulation of the antibodymay be prepared in a pH-buffered solution, for example, at pH rangingfrom about 4.5 to about 6.5, or from about 4.8 to about 5.5, oralternatively about 5.0. Examples of buffers that are suitable for a pHwithin this range include acetate (e.g. sodium acetate), succinate (suchas sodium succinate), gluconate, histidine, citrate and other organicacid buffers. The buffer concentration can be from about 1 mM to about200 mM, or from about 10 mM to about 60 mM, depending, for example, onthe buffer and the desired isotonicity of the formulation.

A tonicity agent, which may also stabilize the antibody, may be includedin the formulation. Exemplary tonicity agents include polyols, such asmannitol, sucrose or trehalose. Preferably the aqueous formulation isisotonic, although hypertonic or hypotonic solutions may be suitable.Exemplary concentrations of the polyol in the formulation may range fromabout 1% to about 15% w/v.

A surfactant may also be added to the antibody formulation to reduceaggregation of the formulated antibody and/or minimize the formation ofparticulates in the formulation and/or reduce adsorption. Exemplarysurfactants include nonionic surfactants such as polysorbates (e.g.polysorbate 20, or polysorbate 80) or poloxamers (e.g. poloxamer 188).Exemplary concentrations of surfactant may range from about 0.001% toabout 0.5%, or from about 0.005% to about 0.2%, or alternatively fromabout 0.004% to about 0.01% w/v.

In one embodiment, the formulation contains the above-identified agents(i.e. antibody, buffer, polyol and surfactant) and is essentially freeof one or more preservatives, such as benzyl alcohol, phenol, m-cresol,chlorobutanol and benzethonium C1. In another embodiment, a preservativemay be included in the formulation, e.g., at concentrations ranging fromabout 0.1% to about 2%, or alternatively from about 0.5% to about 1%.One or more other pharmaceutically acceptable carriers, excipients orstabilizers such as those described in Remington's PharmaceuticalSciences 16th edition, Osol, A. Ed. (1980) may be included in theformulation provided that they do not adversely affect the desiredcharacteristics of the formulation. Acceptable carriers, excipients orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed and include; additional buffering agents; co-solvents;antoxidants including ascorbic acid and methionine; chelating agentssuch as EDTA; metal complexes (e.g. Zn-protein complexes); biodegradablepolymers such as polyesters; and/or salt-forming counterions such assodium.

Therapeutic formulations of the antibody are prepared for storage bymixing the antibody having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose,maltose, or dextrins; chelating agents such as EDTA; sugars such assucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions suchas sodium; metal complexes (e.g., Zn-protein complexes); and/ornon-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol(PEG).

In one embodiment, a suitable formulation of the claimed inventioncontains an isotonic buffer such as a phosphate, acetate, or TRIS bufferin combination with a tonicity agent such as a polyol, Sorbitol, sucroseor sodium chloride which tonicifies and stabilizes. One example of sucha tonicity agent is 5% Sorbitol or sucrose. In addition, the formulationcould optionally include a surfactant such as to prevent aggregation andfor stabilization at 0.01 to 0.02% wt/vol. The pH of the formulation mayrange from 4.5-6.5 or 4.5 to 5.5. Other exemplary descriptions ofpharmaceutical formulations for antibodies may be found in US2003/0113316 and U.S. Pat. No. 6,171,586, each incorporated herein byreference in its entirety.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to further provide an immunosuppressiveagent. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Suspensions and crystal forms of antibodies are also contemplated.Methods to make suspensions and crystal forms are known to one of skillin the art.

The formulations to be used for in vivo administration must be sterile.The compositions of the invention may be sterilized by conventional,well known sterilization techniques. For example, sterilization isreadily accomplished by filtration through sterile filtration membranes.The resulting solutions may be packaged for use or filtered underaseptic conditions and lyophilized, the lyophilized preparation beingcombined with a sterile solution prior to administration.

The process of freeze-drying is often employed to stabilize polypeptidesfor long-term storage, particularly when the polypeptide is relativelyunstable in liquid compositions. A lyophilization cycle is usuallycomposed of three steps: freezing, primary drying, and secondary drying;Williams and Polli, Journal of Parenteral Science and Technology, Volume38, Number 2, pages 48-59 (1984). In the freezing step, the solution iscooled until it is adequately frozen. Bulk water in the solution formsice at this stage. The ice sublimes in the primary drying stage, whichis conducted by reducing chamber pressure below the vapor pressure ofthe ice, using a vacuum. Finally, sorbed or bound water is removed atthe secondary drying stage under reduced chamber pressure and anelevated shelf temperature. The process produces a material known as alyophilized cake. Thereafter the cake can be reconstituted prior to use.

The standard reconstitution practice for lyophilized material is to addback a volume of pure water (typically equivalent to the volume removedduring lyophilization), although dilute solutions of antibacterialagents are sometimes used in the production of pharmaceuticals forparenteral administration; Chen, Drug Development and IndustrialPharmacy, Volume 18, Numbers 11 and 12, pages 1311-1354 (1992).

Excipients have been noted in some cases to act as stabilizers forfreeze-dried products; Carpenter et al., Developments in BiologicalStandardization, Volume 74, pages 225-239 (1991). For example, knownexcipients include polyols (including mannitol, sorbitol and glycerol);sugars (including glucose and sucrose); and amino acids (includingalanine, glycine and glutamic acid).

In addition, polyols and sugars are also often used to protectpolypeptides from freezing and drying-induced damage and to enhance thestability during storage in the dried state. In general, sugars, inparticular disaccharides, are effective in both the freeze-dryingprocess and during storage. Other classes of molecules, including mono-and di-saccharides and polymers such as PVP, have also been reported asstabilizers of lyophilized products.

For injection, the pharmaceutical formulation and/or medicament may be apowder suitable for reconstitution with an appropriate solution asdescribed above. Examples of these include, but are not limited to,freeze dried, rotary dried or spray dried powders, amorphous powders,granules, precipitates, or particulates. For injection, the formulationsmay optionally contain stabilizers, pH modifiers, surfactants,bioavailability modifiers and combinations of these.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsule. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and yethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the Lupron Depot™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

The formulations of the invention may be designed to be short-acting,fast-releasing, long-acting, or sustained-releasing as described herein.Thus, the pharmaceutical formulations may also be formulated forcontrolled release or for slow release.

Specific dosages may be adjusted depending on conditions of disease, theage, body weight, general health conditions, sex, and diet of thesubject, dose intervals, administration routes, excretion rate, andcombinations of drugs. Any of the above dosage forms containingeffective amounts are well within the bounds of routine experimentationand therefore, well within the scope of the instant invention.

The specific binding agent or antibody is administered by any suitablemeans, including parenteral, subcutaneous, intraperitoneal,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions include intravenous,intraarterial, intraperitoneal, intramuscular, intradermal orsubcutaneous administration. In addition, the specific binding agent orantibody is suitably administered by pulse infusion, particularly withdeclining doses of the specific binding agent or antibody. Preferablythe dosing is given by injections, most preferably intravenous orsubcutaneous injections, depending in part on whether the administrationis brief or chronic. Other administration methods are contemplated,including topical, particularly transdermal, transmucosal, rectal, oralor local administration e.g. through a catheter placed close to thedesired site. Most preferably, the specific binding agent or antibody ofthe invention is administered intravenously in a physiological solutionat a dose ranging between 0.01 mg/kg to 100 mg/kg at a frequency rangingfrom daily to weekly to monthly (e.g. every day, every other day, everythird day, or 2, 3, 4, 5, or 6 times per week), preferably a doseranging from 0.1 to 45 mg/kg, 0.1 to 15 mg/kg or 0.1 to 10 mg/kg at afrequency of 2 or 3 times per week, or up to 45 mg/kg once a month.

Administration with Other Agents

The antibodies of the invention also may be concurrently administeredwith other anti-inflammatory therapeutic agents. Concurrentadministration includes administration of the two different therapeuticagents at different times and at different routes, as long as there issome overlap in the time during which the agents are exerting theirtherapeutic effects. Exemplary anti-c-Kit agents known in the artinclude Imatinib Mesylate (Gleevec™). It should be noted that ImatinibMesylate also antagonizes signaling from the Abl tyrosine kinase andtherefore is not a specific c-Kit inhibitor.

EXAMPLES Humanization of SR-1

SR-1 was humanized by a straight CDR graft, with surprisingly no backmutations required to maintain affinity though it remained to bedemonstrated desired functional activity. The human frameworks thatmaintained the most canonical residues, and did not introduce additionalproline residues, were chosen as acceptor sequences. Based on thesecriteria, the heavy chain acceptor sequence was VH1 1-46 for framework Iand II and VH1 1-e for framework III, with JH4 as the closest J region(also known as framework IV). The light chain acceptor sequence was theVK4 B3 germline sequence with JK2 as the closest J region.

Isotype switching was done to produce human IgG2, IgG1, IgG4P andaglycosylated IgG1 forms of the humanized antibody. The N-linkedglycosylation consensus site was removed from the human IgG1 constantregion sequence by mutation of a single residue from asparagine toglutamic acid at position 297 (Kabat numbering).

The humanized SR-1 in the aglycosylated IgG1 (hSR-1 aIgG1) form binds ina desired manner with higher affinity to the membrane c-Kit compared tosoluble c-kit, and is a highly potent neutral antagonist of SCF andmediating no agonism of c-Kit directly in all cell based assays testedusing proximal and distal read-outs of c-Kit signaling. Theaglycosylated IgG1 isotype was chosen to avoid effector function andcell killing through bystander effects. This antibody showed anunexpected and desired half-life, nonlinear PK and saturabletarget-mediated antibody elimination in monkeys. It also depletes mastcells in vivo as expected.

Binding to c-Kit Dimer

Activation of c-Kit upon binding by stem cell factor (SCF) leads todimerization/oligomerization, autophosphorylation and receptorinternalization, most likely through a clathrin-dependent pathway. TheSR-1 monoclonal antibody binds the c-Kit dimer with 1000-fold higheraffinity compared to the soluble c-Kit extra-cellular domain monomer asdetermined by Biacore. Kinetic modeling suggests that SR-1 wouldpreferentially bind the native membrane-associated receptor even in thepresence of ng/mL of the soluble shed receptor monomer.

Carbohydrates present on a glycoprotein can influence biological andfunctional properties. Humanization of SR-1 to an aglycosylated IgG1form showed that binding parameters were conserved. Humanized SR-1 aIgG1bound to recombinant c-Kit receptor-Fc with a KinExA Equilibrium BindingKd of 1.0 pM and using the Biacore assay, hSR-1 aIgG1 blocked stem cellfactor (SCF) binding with a Ki=70 pM. hSR-1 aIgG1 binds with highaffinity to the receptor dimer versus monomer. This is an importantcharacteristic not predicted to be translated with certainty with thehumanization and as the soluble c-Kit monomer is less likely to act as asink for the antibody in vivo.

Inhibition of c-Kit Dependent Cell Survival and Receptor Signaling

The human megakaryoblastic cell line UT-7 is dependent on SCF forsurvival and the removal of SCF or its inhibition results in rapid lossof viability and decreased proliferation. This assay is suited for IC50potency determination of SCF antagonists. hSR-1 aIgG1 exhibited a meanIC50 of 35 pM.

hSR-1 aIgG1 potently inhibited SCF-mediated c-Kit phosphorylation andinternalization in MO7e cells indicating that the antibody can blockSCF-mediated c-Kit signaling events. In contrast to findings of SR-1being capable of intrinsically mediating c-Kit internalization andphosphorylation, surprisingly, no evidence of agonism was detected inthe MO7e c-Kit receptor proximal phosphorylation read-out for hSR-1aIgG1. Notably, hSR-1 IgG2, the IgG2 antibody was slightly less potentand did not fully inhibit SCF-mediated c-Kit receptor internalization.

hSR-1 aIgG1 shows neutralization at 1.0 ug/mL of the synergistic effectof SCF on GM-CSF derived colony formation using primary isolated humanCD34+, CD 117+ (c-Kit) bone marrow cells. Consistent with our novelfinding that hSR-1 aIgG1 did not mediate c-Kit internalization orphosphorylation, and no intrinsic agonistic survival activity of hSR-1aIgG1 was observed up to 10 ug/mL concentration of the antibody in thisassay. In fact, the antibody was able to inhibit survival belowbaseline.

Lack of Mast Cell Aggregation, CDC and FcR Activity

Cultured human mast cells derived from bone marrow CD34+ cells were usedto assess the apparent potency and rank order of compounds. hSR-1 aIgG1inhibited SCF-dependent mast cell survival, conferred no survival signalto mast cells, did not mediate c-Kit receptor phosphorylation (FIG. 3)and showed no ability to mediate homotypic mast cell aggregation. Incontrast, the hSR-1 IgG2 was able to block SCF-mast cell survival butitself showed partial agonist activity conferring a survival signal,mediating c-Kit receptor phosphorylation and resulted in a reproducibleeffect on mast cell clustering. No unexpected abnormalities wereobserved for hSR-1 aIGg1 when this antibody was dosed in vivo up to 30mg/kg once weekly for 4 weeks or up to 150 mg/kg subcutaneously onceweekly for 2 weeks in non-human primates.

hSR-1 aIgG1 shows no detectable non-specific FcR binding to U-937 cellsexpressing Fcγ receptor I=CD64, Fcγ receptor II=CD32 and Fcγ receptorIII=CD16. In contrast, binding of SR-1 IgG1 and IgG4β isotypes wasdetected presumably to the high affinity FcγRI. Therefore no ADCCactivity is predicted for hSR-1 aIgG1, and experimental data to dateshow no complement dependent cytotoxic cell death. Aglycosylatedchimeric mouse/human IgG1 antibodies have been reported to retain someeffector function (Hybridoma. 1991 April; 10(2):211-7) and so thesedesired activities of hSR-1 aIgG1 are not expected. The data shows thatapplication of standard methodologies and therefore choice of typicalIgG2 or IgG1 or IgG4 isotypes would have not yielded a molecule withappropriate characteristics, that is a high-affinity binder, functionalneutral antagonist at c-kit and that would not activate mast cells.

Pharmacokinetics

A preliminary PK study was conducted to compare hSR-1 IgG2 and hSR-1aIgG1 PK in male cynomolgus monkeys after a single IV or SCadministration at 3 mg/kg. Time profiles indicate a non-linear PK forboth. The concentrations decreased more rapidly at lower concentrations.The two antibodies showed similar exposures, as measured by C₀/C_(max)and AUC_(0-tlast), after a single IV or SC administration in cynomolgusmonkeys. Based on AUC_(0-tlast), the serum clearance was approximately≦0.3 mL/hr/kg for both humanized antibodies. The bioavailability wasapproximately 82% and 69% for the hSR-1 aIgG1 and hSR-1 IgG2 humanizedSR-1 versions, respectively, after SC dosing.

Based on preliminary exposure data of SR-1 and humanized antibodies inAfrican green monkeys after repeated once-weekly dosing, humanizedantibodies achieved higher exposures compared to SR-1. Notably,hSR-aIgG1 showed equal best in group PK and it has been demonstratedpreviously that the degree of glycosylation of a molecule may alter itspharmacokinetic properties and, in the case of an antibody, itsmetabolism and other biological properties Cancer Immunol Immunother.1992; 35(3): 165-74.

Table 1. Pharmacokinetic Parameter Estimates After a Single IV or SCAdministration of hSR-1 IgG2 or hSR-1 aIgG1 at 3 mg/kg to MaleCynomolgus Monkeys

TABLE 1 Pharmacokinetic Parameter Estimates After a Single IV or SCAdministration of hSR-1 IgG2 or hSR-1 aIgG1 at 3 mg/kg to MaleCynomolgus Monkeys Dose C₀/C_(max) T_(max) AUC_(0-tlast) CL or CL/F^(a)Test article route (μg/mL) (hr) (hr * μg/mL) (mL/hr/kg) F % hSR-1 aIgG1IV 103 — 9710 ≦0.309 — hSR-1 IgG2 IV 107 — 10600 ≦0.283 — hSR-1 aIgG1 SC36.4 72 7970 ≦0.376 82.1 hSR-1 IgG2 SC 36.7 60 7290 ≦0.412 68.8 C₀ =estimated initial concentration after IV dosing C_(max) = maximumconcentration after SC dosing T_(max) = time of C_(max) AUC_(0-tlast) =area under the concentration-time curve from time 0 to the last timepoint with a quantifiable concentration CL = clearance after IV dosing;CL/F = apparent clearance after SC dosing F % = bioavailability %^(a)Clearance calculated based on AUC_(0-tlast) — not applicable C₀,C_(max), AUC_(0-{last}), CL, CL/F, and F % reported to 3 significantFIGURES.C₀=estimated initial concentration after IV dosingC_(max)=maximum concentration after SC dosingT_(max)=time of C_(max)AUC_(0-tlast)=area under the concentration-time curve from time 0 to thelast time point with a quantifiable concentrationCL=clearance after IV dosing; CL/F=apparent clearance after SC dosingF %=bioavailability %^(a)Clearance calculated based on AUC_(0-tlast)- not applicableC₀, C_(max), AUC_(0-{last}), CL, CL/F, and F % reported to 3 significantFigures.

Human Dose Projections

The minimal effective dose in the wound PD model of mast cell expansionis <0.3 mg/Kg administered once weekly for 2 weeks in monkeys. Based ona body surface area-based dose conversion, the minimal effective dose inhuman is projected to be <0.1 mg/Kg with an equivalent dosing regiment.However, this is a preliminary estimation as the PK and thepharmacodynamic relationship between the degree and duration of c-kitinhibition in human by hSR-1 aIgG1 and clinical endpoints are unknown atthis time. A more accurate projection will be made when morepharmacokinetic and pharmacodynamic data are available.

In Vivo Potency: Depletion of Basal Lung and Colon Mast Cells in Monkeywith SR-1 and hSR-1 aIGg1

In human, mast cell MCt expressing tryptase and lacking chymase arelocalized primarily to mucosal tissues such as the lung and colon, andthis subtype has been detected in the skin and at higher levels of somescleroderma patients suggesting possible alternative activation of mastcells in this condition. The mast cell MCtc expressing both tryptase andchymase are also colocalized in some of these tissues and similarly havebeen associated with scleroderma and other fibrotic conditions. Henceboth subtypes would represent the primary targets for a c-Kit inhibitorin diseases involving mucosal and connective tissues (eg. IPF, SSc,asthma, RA and IBD). The therapeutic would also need to be highlypotent, efficacious and have a good volume of distribution and PK sincemast cells are generally long-lived and are tissue-resident. Moreovermast cells are largely quiescent until activated to degranulate and denovo synthesize mediators where they then play a key role in theinflammatory response.

The aims for the in vivo studies were to demonstrate depletion of basalmucosal and connective tissue mast cells such as present in the lung andcolon and to determine effects on hematopoiesis and effects on precursorcells as well as an impact on erythropoiesis, melanogenesis andspermatogenesis (therefore utility in male contraception) followingsustained and high fractional inhibition of c-Kit. The SR-1 monoclonalantibody was selected based on its equivalent functional potency athuman and monkey c-Kit in the CD34+ bone marrow cell CFU assay(inhibition at 1.0 ug/mL), and its monkey PK.

SR-1 was administered at doses ranging from 3 mg/Kg to 30 mg/Kg onceweekly for 4 weeks. In time course studies, basal colon mast cells wereshown to be maximally depleted after 2 doses by day 14(C_(trough)>800-fold the cell IC50), and thus day 14 was chosen as thetime point to determine the pharmacological activity of c-Kitantagonists on basal colon mast cells. For practical reasons, basalpulmonary mast cells were evaluated on day 28 at the time of necropsyand the termination of the study.

At a 3.0 mg/Kg dose of SR-1 administered once weekly, depletion of basallung mast cells was observed at a C_(trough) PK level of >200-fold theUT-7 cellular IC50. The effects of lower doses of SR-1 on basal colonand lung mast cells, melanogenesis and spermatogenesis were notevaluated.

However, a lower dose study with hSR-1 aIgG1 was performed at 0.3, 1.0and 3.0 mg/Kg. The day 14 C_(trough) levels were >200, >2000,and >8000-fold the cell IC50 and these C_(trough) levels corresponded tono efficacy, near half-depletion (69%), and near-full depletion (96%) ofbasal colon mast cells (summarized in Table 1). The exposure, cellpotency and effect relationship for hSR-1 aIgG1 is in correspondencewith SR-1 findings reported.

In Vivo Efficacy of SR-1 and hSR-1 aIGg1 in the Wound PharmacodynamicModel of Mast Cell Expansion in Monkey

Damage to the skin is followed by a robust inflammatory response, inwhich first neutrophils and then macrophages and mast cells emigratefrom nearby tissues and from the circulation, granulation and,re-epithelialization of tissues, and fibroblast associated contractionof underlying wound connective tissues (Diegelmann R F, et al., Front.Biosci. 2004, January 1; 9:283-9). Cutaneous wound injury is a model tostudy mechanisms that may be relevant in fibrosis since many of the celltypes involved are associated with this disease. Moreover, it has beenreported in humans to be coupled with a rise in fibroblast derived SCFand activation and increased densities of mast cells (Trautmann A, etal, J. Pathol. 2000, January; 190(1):100-6). Following cutaneous woundinjury in monkey, mast cell numbers increase in a time-dependent mannerwith a plateau that is reached 14-days post-wounding, which iscomparable to the human paradigm.

Doses of 0.3, 1 or 3 mg/Kg of SR-1 administered once weekly led to nearmaximal inhibition of wound activated expansion of mast cells on day 14(FIG. 1). Maximal inhibition is defined as the ability to block 100% theincrease in mast cells over baseline numbers by day 14 after wounding.C_(trough) levels for the 0.3 mpk dose was >7-fold the UT-7 IC50 on Day14 (Table 2). By 3 weeks, serum levels were about 2-fold the UT-7 IC50,and at this exposure only partial inhibition was observed (FIG. 1). At 3weeks, maximal efficacy was still observed for both the 1 and 3 mg/Kgcohorts where serum C_(trough) levels were sustained at >200-fold theIC50. These studies suggest that a sustained C_(trough) exposureof >7-fold the IC50 concentration is likely required for maximalinhibition of wound-expanded mast cells.

Doses of 0.3, 1.0 and 3.0 mg/Kg of hSR-1 aIgG1 were evaluated based onthe maximal efficacy shown for SR-1 in this model. At the lowest dosetested (0.3 mg/Kg), maximal inhibition of wound-induced expansion ofmast cells was observed within 2 weeks. Serum C_(trough) levels at thistime was >200-fold the UT-7 IC50 (Table 2).

Table 2 summarizes the PD/PK effects of SR-1 and hSR-1 aIGg1 in thewound PD model.

Inhibition of Depletion UT-7 Fold activated of basal IC50 C_(trough)UT-7 skin mast colon drug (ng/ml) Dose (ng/ml) IC50 cells mast cellsSR-1 3.6 0.3 mg/kg 30 8 >95% ND 1.0 mg/kg 994 276 >80% ND 3.0 mg/kg 1873520 >80% ND hSR-1 4.5 0.3 mg/kg 910 >200 >95% No effect aIgG1 1.0 mg/kg12,400 >2000 >95% >65% 3.0 mg/kg 44,500 >7000 >95% >95%

An incisional wound was made followed by punch biopsy up to day 21 inhuman (left) or non-human primate (right) (FIG. 2). Mast cells and/orSCF expressing fibroblasts were revealed by chromogenic stain or IHCrespectively. In human, the expression of SCF rises and returns tobaseline and is temporally followed by a transient rise in mast cellnumbers during normal wound healing. Similar mast cell response towounding is observed in monkey. During fibrosis and aberrant woundhealing, SCF expression and mast cells numbers remain elevated (FIG. 2).

Hematopoiesis and Melanogenesis

Mouse genetics show that c-Kit plays a role in hematopoiesis duringembryonic development, but in human heterozygous inactivating and/orloss-of-function c-Kit mutations in Piebald subjects have not beenlinked to hematological abnormalities. SCF and c-Kit are important inhuman hematopoiesis since SCF is used in combination with G-CSF forhematopoietic stem cell mobilization. Moreover, the multi-kinaseinhibitor Gleevec which target primarily BCR-ABL, PDGFR and c-Kit has asits primary pharmacological effect myelosuppression, and severe grade3-4 anemias and thrombocytopenias have been reported in GIST patients(Hensley M L, et al., Semin. Hematol. 2003, April; 40(2 Suppl 2):21-5).

Mouse genetics indicate that c-Kit plays a role in the migration ofmelanoblasts from the neural crest during embroyogenesis, and this roleis supported in human Piebaldism. Gleevec has been reported to cause“banding” depigmentation in the hair in a minor number of GIST patientsbut this has not been a consistent finding and hyperpigmentation hasalso been reported. The contribution of other kinases such as PDGF cannot be excluded. Studies in mice with multi-kinase inhibitors and ac-Kit antibody show that inhibition of hair pigmentation is fullyreversible suggesting that c-Kit inhibition affects melanocyte functionand not survival in the post-natal setting (Moss et al, 2003).

SR-1 was used in dose-ranging studies from 3 to 30 mg/Kg administeredonce weekly for 4 weeks to determine the exposures required to inhibithematopoiesis, spermatogenesis and active melanogenesis. A full bloodpanel including cell differentials was performed on blood samples takenat baseline, day 4, 7, 14, 21 and 28 after the start of the study.Analysis of freshly isolated blood samples was performed at the QueenElizabeth Hospital Clinical Hematology lab, Barbados.

No significant impact of SR-1 compared to control subjects and baselinevalues was detected on any hematological parameters analyzed thoughthere were non-significant decreases in RBCs in drug treated animals 4weeks after dosing started. At the highest dose tested, 30 mg/Kg onceweekly over 4 weeks, exposure levels of >70.000-fold the UT-7 IC50potency was achieved. Lack of significant effect on hematologicalparameters were confirmed by bone marrow histopathological analysisshowing no difference between drug treated and control cohorts and nodepletion of CD117 positive hematopoietic stem cells suggestingpotential redundant pathways for hematopoiesis in the African greenmonkey NHP species. The effect of 3-30 mpk administered once weekly for4 weeks on melanogenesis was also examined since there may be utility inmelanoma. To assess the effects on activated melanocytes which maybetter reflect a disease state, hair was depilated to activatemelanogenesis. The normal hair color of the coat was not visiblyaffected in any cohort. However, inhibition of hair pigmentation tovariable degrees was observed in newly regrown hair in monkeys receivingthe 30 mg/Kg dose. No effect was observed in the 10 mg/Kg cohortsuggesting that the no effect dose is between 10-30 mg/Kg. In the 10mg/Kg cohort, SR-1 exposures were >8000-fold the UT-7 IC50. Maximalefficacy for mast cell depletion was achieved at >7-fold the UT-7 IC50.These data suggests that c-Kit inhibition affects melanocyte function,and that higher doses/exposures may be needed to block in diseasescharacterized by excessive melanocyte activity.

Spermatogenesis

Mouse studies have shown that c-Kit is important for the maintenance andproliferation of differentiated c-kit receptor-positive spermatogoniabut not for the initial step of spermatogonial cell differentiation.Both male and female Piebald subjects that are heterozygote for aninactive c-Kit receptor allele, are fertile suggesting that this degreeof c-Kit inactivation, does not appear to affect primordial germ celldevelopment, spermatogenesis or oogenesis.

SR-1 showed dose-dependent inhibition of spermatogenesis from 0.3-30mg/Kg. The 0.3 mg/Kg once weekly dose is less than the maximal effectobserved at the higher doses, and lower dose ranging studies arerequired to define the ED50. The exposures achieved are 7-fold the UT-7IC50 which is the exposure required for maximal efficacy in the woundmodel of mast cell expansion. PK extrapolation suggested that theantibody may likely clear 1 month after the last dose, but 9 months wasselected as a conservative time point to assess recovery. Normalspermatogenesis was found in all dosed animals at 9 months demonstratingthat use of a hSR-1 aIgG1-like molecule is useful as a malecontraceptive.

Summary

Humanized anti-c-Kit aglycosylated IgG1 (hSR-1 aIgG1) antibody is ahighly potent and specific antibody that is neutralizing in all cellbased assays tested using proximal and distal read-outs of c-Kitsignaling. Intrinsically it does not mediate c-Kit receptorinternalization or phosphorylation as reported for the parent murinemonoclonal SR-1 antibody. The selection of the aglycosylated IgG1isotype over humanized IgG1, IgG2 and IgG4 isotypes would not have beenpredicted based on standard approaches. hSR-1 aIgG1 was chosenempirically via novel experimentation to show that it exhibited theappropriate pharmacological characteristics at the membrane c-Kitreceptor, avoid agonist activity at c-Kit and on mast cells, lackingeffector function and cell killing through bystander effects. Thisantibody showed good s.c. bioavailability and half-life, nonlinear PKand saturable target-mediated antibody elimination and mast celldepletion in monkeys. These data would predict a suitable humanefficacious mast cell depleting dose.

The minimal efficacious dose of the parent mouse SR-1 monoclonal in themonkey wound PD model is <0.3 mg/Kg (C_(trough)>7-fold the cell IC50),and at slightly higher exposure (C_(trough)>800-fold the cell IC50) wasefficacious as well in depleting basal skin, colon and lung mast cells.Similarly, exposure levels greater than >8000-fold the cell IC50 isrequired before qualitative impacts on hair pigmentation in newlyregrown hair can be observed. Inhibition of hair pigmentation in newlygrowing hair has been reported with multi-kinase inhibitors and a c-Kitantibody in rodent and the hSR-1 aIgG1 may have utility in diseasesassociated with excessive melanocyte activity. The effect is reversibleupon cessation of treatment. A sub-maximal effect on inhibition ofspermatogenesis was shown at levels >7-fold the cell IC50, the exposurethat conferred maximal efficacy in the wound PD model.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, in their entirety.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention.

1.-19. (canceled)
 20. A nucleic acid encoding a specific binding agentthat binds c-Kit comprising an amino acid sequence at least 80%identical to an amino acid sequence selected from the group consistingof those set forth in SEQ ID NOS: 2, 4 or
 6. 21. A nucleic acidcomprising a nucleic acid sequence at least 90% identical to a nucleicacid selected from the group set forth in SEQ ID NOS: 1, 3, or
 5. 22. Avector comprising the nucleic acid of claim
 21. 23. A host cellcomprising the vector of claim
 22. 24. A method of producing a specificc-Kit binding agent comprising culturing a host cell of claim 23 suchthat the nucleic acid is expressed to produce the specific bindingagent.
 25. The method of claim 24, further comprising the step ofrecovering the specific binding agent from the host cell culture.26.-32. (canceled)